Cycloalkyl-[4-(difluorophenyl)-oxazol-5-yl]-triazolo-pyridines

ABSTRACT

The present invention relates to novel cycloalkyl-[4-(difluorophenyl)-oxazol-5-yl]triazolo-pyridines, to intermediates for their preparation, to pharmaceutical compositions containing them and to their medicinal use. The compounds of the present invention are potent inhibitors of MAP kinases, preferably p38 kinase. They are useful in the treatment of inflammation, osteoarthritis, rheumatoid arthritis, cancer, reperfusion or ischemia in stroke or heart attack, autoimmune diseases and other disorders.

[0001] The present invention relates to novelcycloalkyl-[4-(difluorophenyl)-oxazol-5-yl]triazolo-pyridines, tointermediates for their preparation, to pharmaceutical compositionscontaining them and to their medicinal use. The compounds of the presentinvention are potent inhibitors of MAP kinases, preferably p38 kinase.They are useful in the treatment of inflammation, osteoarthritis,rheumatoid arthritis, cancer, reperfusion or ischemia in stroke or heartattack, autoimmune diseases and other disorders.

[0002] Intracellular signal transduction is the means by which cellsrespond to extracellular stimuli. Regardless of the nature of the cellsurface receptor (e.g. protein tyrosine kinase or seven-transmembraneG-protein coupled), protein kinases and phosphatases along withphospholipases are the essential machinery by which the signal isfurther transmitted within the cell [Marshall, J. C., Cell, 80, 179-278(1995)]. Protein kinases can be categorized into five classes with thetwo major classes being, tyrosine kinases and serine/threonine kinasesdepending upon whether the enzyme phosphorylates its substrate(s) onspecific tyrosine(s) or serine/threonine(s) residues [Hunter, T.,Methods in Enzymology, (Protein Kinase Classification) p. 3, Hunter, T.;Sefton, B. M.; eds. vol. 200, Academic Press; San Diego, 1991].

[0003] For most biological responses, multiple intracellular kinases areinvolved and an individual kinase can be involved in more than onesignaling pathway. These kinases are often cytosolic and can translocateto the nucleus or the ribosomes where they can affect transcriptionaland translational events, respectively. The involvement of kinases intranscriptional control is presently much better understood than theireffect on translation as illustrated by the studies on growth factorinduced signal transduction involving MAP/ERK kinase [Marshall, C. J.,Cell, 80, 179 (1995); Herskowitz, I., Cell, 80, 187 (1995); Hunter, T.,Cell, 80, 225 (1995); Seger, R., and Krebs, E. G., FASEB J., 726-735(1995)].

[0004] While many signaling pathways are part of normal cellhomeostasis, numerous cytokines (e.g., IL-1 and TNF) and certain othermediators of inflammation (e.g., COX-2, and iNOS) are produced only as aresponse to stress signals such as bacterial lipopolysaccharide (LPS).Early evidence suggesting that the signal transduction pathway leadingto LPS-induced cytokine biosynthesis involved protein kinases came fromstudies of Weinstein [Weinstein, et al., J. Immunol., 151, 3829(1993)]but the specific protein kinases involved were not identified. Workingfrom a similar perspective, Han [Han, et al., Science, 265, 808(1994)]identified murine p38 as a kinase which is tyrosine phosphorylated inresponse to LPS. Additional evidence of the involvement of the p38kinase in LPS-stimulated signal transduction pathway leading to theinitiation of proinflammatory cytokine biosynthesis was provided by thediscovery of p38 kinase (MAPK14, CSBP 1 and 2) by Lee [Lee; et al.,Nature, 372, 739(1994)] as the molecular target for a novel class ofanti-inflammatory agents. Thus, compounds which inhibit p38 will inhibitIL-1 and TNF synthesis in human monocytes. Such results have beenreported by [Lee, et al., Int. J. Immunopharmac., 10(7), 835(1988)] and[Lee; et al., Annals N.Y. Acad. Sci., 696, 149(1993)].

[0005] It is now accepted that CSBP/p38 is one of several kinasesinvolved in a stress-response signal transduction pathway which isparallel to and largely independent of the analogous mitogen-activatedprotein kinase (MAP) kinase cascade. Stress signals, including LPS,pro-inflammatory cytokines, oxidants, UV light and osmotic stress,activate kinases upstream from CSBP/p38 which in turn phosphorylateCSBP/p38 at threonine 180 and tyrosine 182 resulting in CSBP/p38activation. MAPKAP kinase-2 and MAPKAP kinase-3 have been identified asdownstream substrates of CSBP/p38 which in turn phosphorylate heat shockprotein Hsp 27. It is now known that MAPKAP-2 is essential for LPSinduced TNFα biosynthesis [Kotlyarov, et al., Nature Cell Biol., 1, 94(1999), see also Cohen, P., Trends Cell Biol., 353-361(1997)].

[0006] In addition to inhibiting IL-1 and TNF, CSBP/p38 kinaseinhibitors also decrease the synthesis of a wide variety ofpro-inflammatory proteins including, IL-6, IL-8, GM-CSF and COX-2.Inhibitors of CSBP/p38 kinase have also been shown to suppress theTNF-induced expression of VCAM-1 on endothelial cells, the TNF-inducedphosphorylation and activation of cytosolic PLA2 and the IL-1 stimulatedsynthesis of collagenase and stromelysin. These and additional datademonstrate that CSBP/p38 is involved not only cytokine synthesis, butalso in cytokine signaling [CSBP/p38 kinase reviewed in Cohen, P.,Trends Cell Biol., 353-361 (1997)].

[0007] Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) arebiological substances produced by a variety of cells, such as monocytesor macrophages. IL-1 has been demonstrated to mediate a variety ofbiological activities thought to be important in immunoregulation andother physiological conditions such as inflammation [See, e.g.,Dinarello, et al., Rev. Infect. Disease, 6, 51 (1984)]. The myriad ofknown biological activities of IL-1 include the activation of T helpercells, induction of fever, stimulation of prostaglandin or collagenaseproduction, neutrophil chemotaxis, induction of acute phase proteins andthe suppression of plasma iron levels.

[0008] There are many disease states in which excessive or unregulatedIL-1 production is implicated in exacerbating and/or causing thedisease. These include rheumatoid arthritis, osteoarthritis, endotoxemiaand/or toxic shock syndrome, other acute or chronic inflammatory diseasestates such as the inflammatory reaction induced by endotoxin orinflammatory bowel disease, tuberculosis, atherosclerosis, muscledegeneration, cachexia, psoriatic arthritis, Reiter's syndrome,rheumatoid arthritis, gout, traumatic arthritis, rubella arthritis, andacute synovitis. Other studies also link IL-1 activity to diabetes andpancreatic 1 cells, Dinarello, J. Clinical Immunology, 5 (5), 287-297(1985).

[0009] Excessive or unregulated TNF production has been implicated inmediating or exacerbating a number of diseases including rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions; sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoidosis, bone resorption diseases, reperfusioninjury, graft vs. host reaction, allograft rejections, fever andmyalgias due to infection, such as influenza, cachexia secondary toinfection or malignancy, cachexia secondary to acquired immunedeficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloidinformation, scar tissue formation, Crohn's disease, ulcerative colitis,or pyrosis.

[0010] Interleukin-8 (IL-8) is a chemotactic factor produced by severalcell types including mononuclear cells, fibroblasts, endothelial cells,and keratinocytes. Its production from endothelial cells is induced byIL-1, TNF, or lipopolysaccharide (LPS). IL-8 stimulates a number offunctions in vitro. It has been shown to have chemoattractant propertiesfor neutrophils, T-lymphocytes, and basophils. In addition it induceshistamine release from basophils from both normal and atopic individualsas well lysosomal enzyme release and respiratory burst from neutrophils.IL-8 has also been shown to increase the surface expression of Mac-1(CD11b/CD18) on neutrophils without de novo protein synthesis, this maycontribute to increased adhesion of the neutrophils to vascularendothelial cells. Many diseases are characterized by massive neutrophilinfiltration. Conditions associated with an increase in IL-8 production(which is responsible for chemotaxis of neutrophils into theinflammatory site) would benefit by compounds which are suppressive ofIL-8 production.

[0011] Human interleukin-18 (IL-18) is another member of the interleukinfamily that has recently been identified. IL-18 is a cytokine that issynthesized as a biologically inactive 193 amino acid precursor protein(Ushio, et al., J. Immunol., 15 6:4274, 1996). Cleavage of the precursorprotein, for example by caspase-1 or caspase-4, liberates the 156 aminoacid mature protein (Gu, et al., Science, 275:206, 1997; Ghayur, et al.,Nature, 386:619, 1997), which exhibits biological activities thatinclude the costimulation of T cell proliferation, the enhancement of NKcell cytotoxicity, the induction of IFN-γ production by T cells and NKcells, and the potentiation of T helper type I (Th I) differentiation(Okamura, et al., Nature, 378:88, 1995; Ushio, et al., J. Immunol.,156:4274, 1996; Micallef, et al., Eur. J. Immunol., 26:1647, 1996;Kohno, et al., J. Immunol., 158:1541, 1997; Zhang, et al., Infect.Immunol., 65:3594, 1997; Robinson, et al., Immunol, 7:571, 1997). Inaddition, IL-18 is an efficacious inducer of human monocyteproinflammatory mediators, including IL-8, tumor necrosis factor-a, andprostaglandin E2 (PGE2) (Ushio, S. et al., J. Immunol., 156:4274-4279,1996; Puren, A. J. et al., J. Clin. Invest., 10:711-721, 1997).

[0012] IL-1 and TNF affect a wide variety of cells and tissues and thesecytokines as well as other leukocyte derived cytokines are important andcritical inflammatory mediators of a wide variety of disease states andconditions. The inhibition of these cytokines is of benefit incontrolling, reducing and alleviating many of these disease states.

[0013] Inhibition of signal transduction via CSBP/p38, which in additionto IL-1, TNF and IL-8 described above is also required for the synthesisand/or action of several additional pro-inflammatory proteins (i.e.,IL-6, GM-CSF, COX-2, collagenase and stromelysin), is expected to be ahighly effective mechanism for regulating the excessive and destructiveactivation of the immune system. This expectation is supported by thepotent and diverse anti-inflammatory activities described for CSBP/p38kinase inhibitors [Badger, et al., J. Pharm. Exp. Thera., 279 (3);1453-1461. (1996); Griswold, et al., Pharmacol. Comm., 7, 323-229(1996)].

[0014] There remains a need for treatment, in this field, for compoundswhich are cytokine suppressive anti-inflammatory drugs, i.e., compoundswhich are capable of inhibiting the MAPK14/CSBP/p38/RK kinase.

[0015] Other kinases differentially affected by the compounds of thepresent invention include: Extracellular signal regulated kinase-1 (ERK1or MAPK3), Extracellular signal regulated kinase-2 (ERK2 or MAPK2),Extracellular signal regulated kinase-3 (ERK3 or MAPK6), Extracellularsignal regulated kinase-5 (ERK5 or MAPK7), Extracellular signalregulated kinase-6 (ERK6 or MAPK12), MAPK1, MAPK4, MAPK8, MAPK9, MAPK10,MAPK11, and MAPK13.

[0016] MAPK14/CSBP/p38/RK kinase inhibitors are well known to thoseskilled in the art. U.S. Provisional Applications 60/274,791, 60/274,840and 60/281,331, filed Mar. 9, 2001, Mar. 9, 2001 and Apr. 4, 2001,respectively, and entitled “Novel Antiinflammatory Compounds,” “NovelTriazolopyridine Antiinflammatory Compounds” and “Novel BenzotriazoleAntiinflammatory Compounds,” respectively, refer to certain inhibitorsof MAP kinases, preferably p38 kinase. International Patent PublicationWO 00/40243, published Jul. 13, 2000, refers to pyridine substitutedpyridine compounds and states that these compounds are p38 inhibitors.International Patent Publication WO 00/63204, published Oct. 26, 2000,refers to substituted azole compounds and states that these compoundsare p38 inhibitors. International Patent Publication WO 00/31065,published Jun. 2, 2000, refers to certain heterocyclic compounds andstates that these compounds are p38 inhibitors. International PatentPublication WO 00/06563, published Feb. 10, 2000, refers to substitutedimidazole compounds and states that these compounds are p38 inhibitors.International Patent Publication WO 00/41698, published Jul. 20, 2000,refers to certain ω-carboxy aryl substituted diphenyl urea compounds andstates that these compounds are p38 inhibitors. U.S. Pat. No. 6,288,062refers to certain substituted oxazole compounds and states that thesecompounds are p38 inhibitors. U.S. Pat. No. 5,716,955 refers to certainsubstituted imidazole compounds and states that these compounds are p38inhibitors. U.S. Pat. No. 5,716,972 refers to certain pyridinylsubstituted imidazole compounds and states that these compounds are p38inhibitors. U.S. Pat. No. 5,756,499 refers to certain substitutedimidazole compounds and states that these compounds are p38 inhibitors.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a compound of the formula

[0018] wherein R¹ is fluoro;

[0019] s is two;

[0020] R² is (C₃-C₆)cycloalkyl optionally substituted by one or twomoieties independently selected from the group consisting of halo,(C₁-C₄)alkyl, hydroxy, (C₁-C₆)alkoxy, and (C₁-C₆)alkyl-(C═O)—O—;

[0021] or pharmaceutically acceptable salts and prodrugs thereof.

[0022] The present invention also relates to the pharmaceuticallyacceptable acid addition salts of compounds of the formula I. The acidswhich are used to prepare the pharmaceutically acceptable acid additionsalts of the aforementioned base compounds of this invention are thosewhich form non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions, such as the chloride, bromide,iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,lactate, citrate, acid citrate, tartrate, bitartrate, succinate,maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)]salts.

[0023] The invention also relates to base addition salts of formula I.The chemical bases that may be used as reagents to preparepharmaceutically acceptable base salts of those compounds of formula Ithat are acidic in nature are those that form non-toxic base salts withsuch compounds. Such non-toxic base salts include, but are not limitedto those derived from such pharmacologically acceptable cations such asalkali metal cations (e.g., potassium and sodium) and alkaline earthmetal cations (e.g., calcium and magnesium), ammonium or watersolubleamine addition salts such as N-methylglucamine-(meglumine), and thelower alkanolammonium and other base salts of pharmaceuticallyacceptable organic amines.

[0024] The compounds of this invention include all stereoisomers (e.g.,cis and trans isomers) and all optical isomers of compounds of theformula I (e.g., R and S enantiomers), as well as racemic,diastereomeric and other mixtures of such isomers.

[0025] The compounds and prodrugs of the present invention can exist inseveral tautomeric forms, including the enol and imine form, the ketoand enamine form and geometric isomers and mixtures thereof. All suchtautomeric forms are included within the scope of the present invention.Tautomers exist as mixtures of tautomers in solution. In solid form,usually one tautomer predominates. Even though one tautomer may bedescribed, the present invention includes all tautomers of the presentcompounds.

[0026] The present invention also includes atropisomers of the presentinvention. Atropisomers refer to compounds of formula I that can beseparated into rotationally restricted isomers.

[0027] The compounds of this invention may contain olefin-like doublebonds. When such bonds are present, the compounds of the invention existas cis and trans configurations and as mixtures thereof.

[0028] As used herein, the term “alkyl,” as well as the alkyl moietiesof other groups referred to herein (e.g., alkoxy), may be linear orbranched (such as methyl, ethyl, n-propyl, isopropyl, n-butyl,iso-butyl, secondary-butyl, tertiary-butyl), optionally substituted by 1to 3 suitable substituents as defined above such as fluoro, chloro,trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy, trifluoromethoxy,difluoromethoxy or (C₁-C₆)alkyl. The phrase “each of said alkyl” as usedherein refers to any of the preceding alkyl moieties within a group suchalkoxy, alkenyl or alkylamino. Preferred alkyls include (C₁-C₄)alkyl,most preferably methyl.

[0029] As used herein, the term “cycloalkyl” refers to a mono orbicyclic carbocyclic ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl,cyclohexenyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl andbicyclo[5.2.0]nonanyl, etc.); optionally containing 1-2 double bonds andoptionally substituted by 1 to 3 suitable substituents as defined abovesuch as fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy,trifluoromethoxy, difluoromethoxy or (C₁-C₆)alkyl. The phrase “each ofsaid alkyl” as used herein refers to any of the preceding alkyl moietieswithin a group such alkoxy, alkenyl or alkylamino. Preferred cycloalkylsinclude cyclobutyl, cyclopentyl and cyclohexyl.

[0030] As used herein, the term “halogen” includes fluoro, chloro, bromoor iodo or fluoride, chloride, bromide or iodide.

[0031] As used herein, the term “halo-substituted alkyl” refers to analkyl radical as described above substituted with one or more halogensincluded, but not limited to, chloromethyl, dichloromethyl,fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trichloroethyl, andthe like; optionally substituted by 1 to 3 suitable substituents asdefined above such as fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy,(C₆-C₁₀)aryloxy, trifluoromethoxy, difluoromethoxy or (C₁-C₆)alkyl.

[0032] As used herein, the term “alkenyl” means straight or branchedchain unsaturated radicals of 2 to 6 carbon atoms, including, but notlimited to ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl,2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like; optionallysubstituted by 1 to 3 suitable substituents as defined above such asfluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy,trifluoromethoxy, difluoromethoxy or (C₁-C₆)alkyl.

[0033] As used herein, the term “(C₂-C₆)alkynyl” is used herein to meanstraight or branched hydrocarbon chain radicals having one triple bondincluding, but not limited to, ethynyl, propynyl, butynyl, and the like;optionally substituted by 1 to 3 suitable substituents as defined abovesuch as fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy,trifluoromethoxy, difluoromethoxy or (C₁-C₆)alkyl.

[0034] As used herein, the term “carbonyl” or “(C═O)” (as used inphrases such as alkylcarbonyl, alkyl-(C═O)— or alkoxycarbonyl) refers tothe joinder of the >C═O moiety to a second moiety such as an alkyl oramino group (i.e. an amido group). Alkoxycarbonylamino (i.e.alkoxy(C═O)—NH—) refers to an alkyl carbamate group. The carbonyl groupis also equivalently defined herein as (C═O). Alkylcarbonylamino refersto groups such as acetamide.

[0035] As used herein, the term “aryl” means aromatic radicals such asphenyl, naphthyl, tetrahydronaphthyl, indanyl and the like; optionallysubstituted by 1 to 3 suitable substituents as defined above such asfluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy,trifluoromethoxy, difluoromethoxy or (C₁-C₆)alkyl.

[0036] As used herein, the term “heteroaryl” refers to an aromaticheterocyclic group usually with one heteroatom selected from O, S and Nin the ring. In addition to said heteroatom, the aromatic group mayoptionally have up to four N atoms in the ring. For example, heteroarylgroup includes pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl,furyl, imidazolyl, pyrrolyl, oxazolyl (e.g., 1,3-oxazolyl,1,2-oxazolyl), thiazolyl (e.g., 1,2-thiazolyl, 1,3-thiazolyl),pyrazolyl, tetrazolyl, triazolyl (e.g., 1,2,3-triazolyl,1,2,4-triazolyl), oxadiazolyl (e.g., 1,2,3-oxadiazolyl), thiadiazolyl(e.g., 1,3,4-thiadiazolyl), quinolyl, isoquinolyl, benzothienyl,benzofuryl, indolyl, and the like; optionally substituted by 1 to 3suitable substituents as defined above such as fluoro, chloro,trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy, trifluoromethoxy,difluoromethoxy or (C₁-C₆)alkyl. Particularly preferred heteroarylgroups include oxazolyl, imidazolyl, pyridyl, thienyl, furyl, thiazolyland pyrazolyl (these heteroaryls are most preferred of the R⁴, R⁵, R⁶and R⁷ heteroaryls).

[0037] The term “heterocyclic” as used herein refers to a cyclic groupcontaining 1-9 carbon atoms and 1-4 hetero atoms selected from N, O, Sor NR′. Examples of such rings include azetidinyl, tetrahydrofuranyl,imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl,thiazolidinyl, pyrazolidinyl, thiomorpholinyl, tetrahydrothiazinyl,tetrahydrothiadiazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl,oxazinyl, oxathiazinyl, indolinyl, isoindolinyl, quinuclidinyl,chromanyl, isochromanyl, benzoxazinyl and the like. Examples of suchmonocyclic saturated or partially saturated ring systems aretetrahydrofuran-2-yl, tetrahydrofuran-3-yl, imidazolidin-1-yl,imidazolidin-2-yl, imidazolidin4-yl, pyrrolidin-1-yl, pyrrolidin-2-yl,pyrrolidin-3-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl,piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, 1,3-oxazolidin-3-yl,isothiazolidine, 1,3-thiazolidin-3-yl, 1,2-pyrazolidin-2-yl,1,3-pyrazolidin-1-yl, thiomorpholinyl, 1,2-tetrahydrothiazin-2-yl,1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morpholinyl,1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, 1,4-oxazin-2-yl,1,2,5-oxathiazin-4-yl and the like; optionally substituted by 1 to 3suitable substituents as defined above such as fluoro, chloro,trifluoromethyl, (C₁-C₆)alkoxy, (C₆-C₁₀)aryloxy, trifluoromethoxy,difluoromethoxy or (C₁-C₆)alkyl. Preferred heterocyclics includetetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl andmorpholinyl.

[0038] More specifically, the present invention also relates to acompound of the formula I wherein R² is optionally substituted(C₃-C₆)cycloalkyl. More specifically, the present invention also relatesto a compound of the formula I wherein R² is cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,cyclopentenyl, cyclohexenyl; optionally containing 1-2 double bonds.More specifically, the present invention also relates to a compound ofthe formula I wherein R² is cyclopropyl or cyclobutyl.

[0039] Another embodiment of the present invention are those compoundsof formula I wherein the compound has the formula

[0040] Another embodiment of the present invention are those compoundsof formula I wherein the compound has the formula

[0041] Another embodiment of the present invention are those compoundsof formula I, wherein R² is (C₃-C₆)cycloalkyl.

[0042] Another embodiment of the invention are those compounds offormula I (or Ia, Ib or Ic), wherein R is (C₃-C₆)cycloalkyl substitutedwith one or two substituents, wherein at least one of said substituentsis halo.

[0043] Another embodiment of the invention are those compounds offormula I (or Ia, Ib or Ic), wherein R² is (C₃-C₆)cycloalkyl substitutedwith one or two substituents wherein at least one of said substituentsis hydroxy, (C₁-C₆)alkyl and (C₁-C₆)alkyl-(C═O)—O—.

[0044] Another embodiment of the present invention are those compoundsof formula I, wherein R² is (C₃-C₆)cycloalkyl substituted with one ortwo (C₁-C₃)alkyl, more specifically one or two methyl, ethyl or propylgroup; more preferably one or two methyl groups.

[0045] Examples of specific preferred difluoro compounds of the formulaI are the following:

[0046]3-Cyclobutyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0047]6-[4-(2,4-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;

[0048]6-[4-(2,5-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;

[0049]3-Cyclopropyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;and

[0050]3-Cyclopropyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine.

[0051] Other specific triazolopyridine compounds of formula I includethe following:

[0052]3-Cyclopentyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0053]3-Cyclohexyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0054]3-Cyclopentyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0055]3-Cyclohexyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0056]3-Cyclopentyl-6-[4-(2,3-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0057]3-Cyclohexyl-6-[4-(2,3-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0058]3-Cyclobutyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;

[0059]6-[4-(2,4-Difluoro-phenyl)-oxazol-5-yl]-3-(1-hydroxy-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;

[0060]6-[4-(2,5-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methoxy-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;

[0061]3-(1-Butyl-cyclopropyl)-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;and

[0062]3-(-Fluoro-cyclopropyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine.

[0063] The present invention also includes isotopically-labelledcompounds, which are identical to those recited in Formula I, but forthe fact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.Compounds of the present invention, prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labelled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labelled compounds of Formula I of this invention andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the Schemes and/or in the Examples andPreparations below, by substituting a readily available isotopicallylabelled reagent for a non-isotopically labelled reagent.

[0064] The compounds of Formula I or a pharmaceutically acceptable saltthereof can be used in the manufacture of a medicament for theprophylactic or therapeutic treatment of any disease state in a human,or other mammal, which is exacerbated or caused by excessive orunregulated cytokine production by such mammal's cells, such as but notlimited to monocytes and/or macrophages.

[0065] Compounds of Formula (I) are capable of inhibitingproinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-18 and TNF andare therefore of use in therapy. IL-1, IL-6, IL-8, IL-18 and TNF affecta wide variety of cells and tissues and these cytokines, as well asother leukocyte-derived cytokines, are important and criticalinflammatory mediators of a wide variety of disease states andconditions. The inhibition of these pro-inflammatory cytokines is ofbenefit in controlling, reducing and alleviating many of these diseasestates.

[0066] Accordingly, the present invention provides a method of treatinga cytokine mediated disease which comprises administering an effectivecytokine-interfering amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof.

[0067] Certain compounds of Formula (I) are capable of inhibitinginducible pro-inflammatory proteins, such as COX-2, also referred to bymany other names such as prostaglandin endoperoxide synthase-2 (PGHS-2)and are therefore of use in therapy. These proinflammatory lipidmediators of the cyclooxygenase (COX) pathway are produced by theinducible COX-2 enzyme. Regulation, therefore of COX-2 which isresponsible for these products derived from arachidonic acid, such asprostaglandins, affect a wide variety of cells and tissues. Expressionof COX-1 is not effected by compounds of Formula (I). This selectiveinhibition of COX-2 is accepted as alleviating or sparing ulcerogenicliability associated with inhibition of COX-1 thereby inhibitingprostoglandins essential for cytoprotective effects. Thus inhibition ofthese pro-inflammatory mediators is of benefit in controlling, reducingand alleviating many of these disease states. Most notably theseinflammatory mediators, in particular prostaglandins, have beenimplicated in pain, such as in the sensitization of pain receptors, oredema. This aspect of pain management, therefore, includes treatment ofneuromuscular pain, headache, cancer pain, and arthritis pain. Compoundsof Formula (I), or a pharmaceutically acceptable salt thereof, are ofuse in therapy in a human, or other mammal, by inhibition of thesynthesis of the COX-2 enzyme.

[0068] Accordingly, the present invention provides a method ofinhibiting the synthesis of COX-2, which comprises administering aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. The present invention also provides for amethod of treatment in a human or other mammal, by inhibition of thesynthesis of the COX-2 enzyme.

[0069] In particular, compounds of Formula (I) or a pharmaceuticallyacceptable salt thereof are of use in the therapy of any disease statein a human, or other mammal, which is exacerbated by or caused byexcessive or unregulated IL-1, IL-8 or TNF production by such mammal'scells, such as, but not limited to, monocytes and/or macrophages.

[0070] Accordingly, in another aspect, this invention relates to amethod of inhibiting the production of IL-1 in a mammal in need thereofwhich comprises administering to said mammal an effective amount of acompound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0071] There are many disease states in which excessive or unregulatedIL-1 production is implicated in exacerbating and/or causing thedisease. These include rheumatoid arthritis, osteoarthritis, meningitis,ischemic and hemorrhagic stroke, neurotrauma/closed head injury, stroke,endotoxemia and/or toxic shock syndrome, other acute or chronicinflammatory disease states such as the inflammatory reaction induced byendotoxin or inflammatory bowel disease, tuberculosis, atherosclerosis,muscle degeneration, multiple sclerosis, cachexia, bone resorption,psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis, gout,traumatic arthritis, rubella arthritis and acute synovitis. Recentevidence also links IL-1 activity to diabetes, pancreatic β cellsdisease, and Alzheimer's disease.

[0072] Use of a p38 inhibitor for the treatment of p38 mediated diseasestates, can include, but is not limited to neurodegenerative diseases,such as Alzheimer's disease, Parkinson's disease and multiple sclerosis,etc. In a further aspect, this invention relates to a method ofinhibiting the production of TNF in a mammal in need thereof whichcomprises administering to said mammal an effective amount of a compoundof Formula (I) or a pharmaceutically acceptable salt thereof.

[0073] Excessive or unregulated TNF production has been implicated inmediating or exacerbating a number of diseases including rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, stroke, cerebral malaria, chronic obstructive pulmonarydisease, chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, bone resorption diseases, such as osteoporosis, cardiac,brain and renal reperfusion injury, graft vs. host reaction, allograftrejections, fever and myalgias due to infection, such as influenza,(including HIV-induced forms), cerebral malaria, meningitis, ischemicand hemorrhagic stroke, cachexia secondary to infection or malignancy,cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS,ARC (AIDS related complex), keloid formation, scar tissue formation,inflammatory bowel disease, Crohn's disease, ulcerative colitis andpyresis.

[0074] Compounds of Formula (I) are also useful in the treatment ofviral infections, where such viruses are sensitive to upregulation byTNF or will elicit TNF production in vivo. The viruses contemplated fortreatment herein are those that produce TNF as a result of infection, orthose which are sensitive to inhibition, such as by decreasedreplication, directly or indirectly, by the TNF inhibiting-compounds ofFormula (I). Such viruses include, but are not limited to HIV-1, HIV-2and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpesgroup of viruses, such as but not limited to, Herpes Zoster and HerpesSimplex. Accordingly, in a further aspect, this invention relates to amethod of treating a mammal afflicted with a human immunodeficiencyvirus (HIV) which comprises administering to such mammal an effectiveTNF inhibiting amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof.

[0075] Compounds of Formula (I) may also be used in association with theveterinary treatment of mammals, other than in humans, in need ofinhibition of TNF production. TNF mediated diseases for treatment, inanimals include disease states such as those noted above, but inparticular viral infections. Examples of such viruses include, but arenot limited to, lentivirus infections such as, equine infectious anaemiavirus, caprine arthritis virus, visna virus, or maedi virus orretrovirus infections, such as but not limited to felineimmunodeficiency virus (FIV), bovine immunodeficiency virus, or canineimmunodeficiency virus or other retroviral infections.

[0076] The compounds of Formula (I) may also be used topically in thetreatment of topical disease states mediated by or exacerbated byexcessive cytokine production, such as by IL-1 or TNF respectively, suchas inflamed joints, eczema, contact dermatitis psoriasis and otherinflammatory skin conditions such as sunburn; inflammatory eyeconditions including conjunctivitis; pyresis, pain and other conditionsassociated with inflammation. Periodontal disease has also beenimplemented in cytokine production, both topically and systemically.Hence, the use of compounds of Formula (I) to control the inflammationassociated with cytokine production in such peroral diseases such asgingivitis and periodontitis is another aspect of the present invention.

[0077] Compounds of Formula (I) have also been shown to inhibit theproduction of IL-8 (Interleukin-8, NAP). Accordingly, in a furtheraspect, this invention relates to a method of inhibiting the productionof IL-8 in a mammal in need thereof which comprises administering, tosaid mammal an effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof.

[0078] There are many disease states in which excessive or unregulatedIL-8 production is implicated in exacerbating and/or causing thedisease. These diseases are characterized by massive neutrophilinfiltration such as, psoriasis, inflammatory bowel disease, asthma,cardiac and renal reperfusion injury, adult respiratory distresssyndrome, thrombosis and glomerulonephritis. All of these diseases areassociated with increased IL-8 production which is responsible for thechemotaxis of neutrophils into the inflammatory site. In contrast toother inflammatory cytokines (IL-1, TNF, and IL-6), IL-8 has the uniqueproperty of promoting neutrophil chemotaxis and activation. Therefore,the inhibition of IL-8 production would lead to a direct reduction inthe neutrophil infiltration.

[0079] The compounds of Formula (I) are administered in an amountsufficient to inhibit a cytokine, in particular IL-1, IL-6, IL-8, IL-18or TNF, production such that it is regulated down to normal levels, orin some case to subnormal levels, so as to ameliorate or prevent thedisease state. Abnormal levels of IL-1, IL-6, IL-8, IL-18 or TNF, forinstance in the context of the present invention, constitute: (i) levelsof free (not cell bound) IL-1, IL-6, IL-8, IL-18 or TNF greater than orequal to 1 picogram per ml; (ii) any cell associated IL-1, IL-6, IL-8,IL-18 or TNF; or (iii) the presence of IL-1, IL-6, IL-8, IL-18 or TNFmRNA above basal levels in cells or tissues in which IL-1, IL-6, IL-8,IL-18 or TNF, respectively, is produced.

[0080] The discovery that the compounds of Formula (I) are inhibitors ofcytokines, specifically IL-1. IL-6, IL-8, IL-18 and TNF is based uponthe effects of the compounds of Formula (I) on the production of theIL-1, IL-8 and TNF in in vitro assays which are described herein or arewell known to those skilled in the art.

[0081] As used herein, the term “inhibiting the production of IL-1(IL-6, IL-8, IL-18 or TNF)” refers to:

[0082] a) a decrease of excessive in vivo levels of the cytokine (IL-1,IL-6, IL-8, IL-18 or TNF) in a human to normal or sub-normal levels byinhibition of the in vivo release of the cytokine by all cells,including but not limited to monocytes or macrophages;

[0083] b) a down regulation, at the genomic level, of excessive in vivolevels of the cytokine (IL-1, IL-6, IL-8, IL-18 or TNF) in a human tonormal or sub-normal levels;

[0084] c) a down regulation, by inhibition of the direct synthesis ofthe cytokine (IL-1, IL-6, IL-8, IL-18 or TNF) or as a postranslationalevent to normal or sub-normal levels; or

[0085] d) a down regulation, at the translational level, of excessive invivo levels of the cytokine (IL-1, IL-6, IL-8, IL-18 or TNF) in a humanto normal or sub-normal levels.

[0086] As used herein, the term “TNF mediated disease or disease state”refers to any and all disease states in which TNF plays a role, eitherby production of TNF itself, or by TNF causing another monokine to bereleased, such as but not limited to IL-1, IL-6, IL-8 or IL-18. Adisease state in which, for instance, IL-1 is a major component, andwhose production or action, is exacerbated or secreted in response toTNF, would therefore be considered a disease state mediated by TNF.

[0087] As used herein, the term “cytokine” refers to any secretedpolypeptide that affects the functions of cells and is a molecule whichmodulates interactions between cells in the immune, inflammatory orhematopoietic response. A cytokine includes, but is not limited to,monokines and lymphokines, regardless of which cells produce them. Forinstance, a monokine is referred to as being produced and secreted by amononuclear cell, such as a macrophage and/or monocyte. Many other cellshowever also produce monokines, such as natural killer cells,fibroblasts, basophils, neutrophils, endothelial cells, brainastrocytes, bone marrow stromal cells, epideral keratinocytes andB-lymphocytes. Lymphokines are generally referred to as being producedby lymphocyte cells. Examples of cytokines include, but are not limitedto Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),Interleukin-18 (IL-18), Tumor Necrosis Factor-alpha (TNF-α) and TumorNecrosis Factor beta (TNF-β).

[0088] As used herein, the term “cytokine interfering” or “cytokinesuppressive amount” refers to an effective amount of a compound ofFormula (I) which will cause a decrease in the in vivo levels of thecytokine to normal or sub-normal levels, when given to a patient for thetreatment of a disease state which is exacerbated by, or caused by,excessive or unregulated cytokine production.

[0089] As used herein, the cytokine referred to in the phrase“inhibition of a cytokine for use in the treatment of a HIV-infectedhuman” is a cytokine which is implicated in (a) the initiation and/ormaintenance of T cell activation and/or activated T cell-mediated HIVgene expression and/or replication and/or (b) any cytokine-mediateddisease associated problem such as cachexia or muscle degeneration.

[0090] As TNF-β (also known as lymphotoxin) has close structuralhomology with TNF-a (also known as cachectin) and since each inducessimilar biologic responses and binds to the same cellular receptor, bothTNF-α and TNF-β are inhibited by the compounds of the present inventionand thus are herein referred to collectively as “TNF” unlessspecifically delineated otherwise.

[0091] A relatively new member of the MAP kinase family, alternativelytermed MAPK14, CSBP, p38 or RK, has been identified by severallaboratories [See Lee, et al., Nature, Vol. 300, n(72), 739-746 (1994)].Activation of this protein kinase via dual phosphorylation has beenobserved in different cell systems upon stimulation by a wide spectrumof stimuli, such as physicochemical stress and treatment withlipopolysaccharide or pro-inflammatory cytokines such as interleukin-1and tumor necrosis factor. The cytokine biosynthesis inhibitors of thepresent invention, compounds of Formula (I), have been determined to bepotent and selective inhibitors of CSBP/p38/RK kinase activity. Theseinhibitors are of aid in determining the signaling pathways involvementin inflammatory responses. In particular, a definitive signaltransduction pathway can be prescribed to the action oflipopolysaccharide in cytokine production in macrophages. In addition tothose diseases already noted herein, treatment of stroke,neurotrauma/CNS head injury, cardiac, brain and renal reperfusioninjury, thrombosis, glomerulonephritis, diabetes and pancreatic β cells,multiple sclerosis, muscle degeneration, eczema, psoriasis, sunburn, andconjunctivitis are also included.

[0092] The cytokine inhibitors were subsequently tested in a number ofanimal models for anti-inflammatory activity. Model systems were chosenthat were relatively insensitive to cyclooxygenase inhibitors in orderto reveal the unique activities of cytokine suppressive agents. Theinhibitors exhibited significant activity in many such in vivo studies.Additionally, the cytokine inhibitors of the present invention areeffective in the collagen-induced arthritis model and inhibition of TNFproduction in the endotoxic shock model. In the latter study, thereduction in plasma level of TNF correlated with survival and protectionfrom endotoxic shock related mortality. Also of great importance are thecompounds' effectiveness in inhibiting bone resorption in a rat fetallong bone organ culture system. Griswold, et al., (1988), ArthritisRheum., 31:1406-1412; Badger, et al., (1989), Circ. Shock, 27, 51-61,Votta, et al., (1994), in vitro. Bone, 15, 533-538; Lee, et al.,(1993.), B Ann. N.Y. Acad. Sci., 696, 149-170.

[0093] It is also recognized that both IL-6 and IL-8 are produced duringrhinovirus (HRV) infections and contribute to the pathogenesis of commoncold and exacerbation of asthma associated with HRV infection (Turner,et al., (1998), Clin. Infec. Dis., Vol. 26, p. 840; Teren, et al.(1997), Am. J. Respir. Crit. Care Med., Vol. 155, p. 1362; Grunberg, etal. (1997), Am. J. ResPir. Crit. Care Med., Vol. 156, p. 609 and Zhu, etal., J. Clin. Invest., (1996), Vol. 97, p 421). It has also beendemonstrated in vitro that infection of pulmonary epithelial cells withHRV results in production of IL-6 and IL-8 (Subauste, et al., J. Clin.Invest., (1995), Vol. 96, p. 549). Epithelial cells represent theprimary site of infection of HRV. Therefore, another aspect of thepresent invention is a method of treatment to reduce inflammationassociated with a rhinovirus infection, not necessarily a direct effectof the virus itself.

[0094] Another aspect of the present invention involves the novel use ofthese p38/cytokine inhibitors for the treatment of chronic inflammatoryor proliferative or angiogenic diseases, which are caused by excessive,or inappropriate angiogenesis.

[0095] Chronic diseases which have an inappropriate angiogenic componentare various ocular neovascularizations, such as diabetic retinopathy andmacular degeneration. Other chronic diseases which have an excessive orincreased proliferation of vasculature are tumor growth and metastasis,atherosclerosis and certain arthritic conditions. Therefore, cytokineinhibitors will be of utility in the blocking of the angiogeniccomponent of these disease states.

[0096] The term “excessive or increased proliferation of vasculatureinappropriate angiogenesis” as used herein includes, but is not limitedto, diseases which are characterized by hemangiomas and ocular diseases.

[0097] The term “inappropriate angiogenesis” as used herein includes,but is not limited to, diseases which are characterized by vesicleproliferation with accompanying tissue proliferation, such as occurs incancer, metastasis, arthritis and atherosclerosis.

[0098] This invention also encompasses methods of treating or preventingdisorders that can be treated or prevented by the inhibition of MAP in amammal, preferably a human, comprising administering to said mammal aneffective amount of a compound of the formula I.

[0099] Accordingly, the present invention provides a method of treatinga p38 kinase mediated disease in a mammal in need thereof, preferably ahuman, which comprises administering to said mammal, an effective amountof a compound of Formula (I) or a pharmaceutically acceptable saltthereof.

[0100] Preferred p38 mediated diseases for treatment include, but arenot limited to psoriatic arthritis, Reiter's syndrome, rheumatoidarthritis, gout, traumatic arthritis, rubella arthritis and acutesynovitis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, Alzheimer's disease, stroke,ischemic and hemorrhagic stroke, neurotrauma/closed head injury, asthma,adult respiratory distress syndrome, chronic obstructive pulmonarydisease, cerebral malaria, meningitis, chronic pulmonary inflammatorydisease, silicosis, pulmonary sarcostosis, bone resorption disease,osteoporosis, restenosis, cardiac reperfusion injury, brain and renalreperfusion injury, chronic renal failure, thrombosis,glomerularonephritis, diabetes, diabetic retinopathy, maculardegeneration, graft vs. host reaction, allograft rejection, inflammatorybowel disease, Crohn's disease, ulcerative colitis, neurodegenerativedisease, multiple sclerosis, muscle degeneration, diabetic retinopathy,macular degeneration, tumor growth and metastasis, angiogenic disease,rhinovirus infection, peroral disease, such as gingivitis andperiodontitis, eczema, contact dermatitis, psoriasis, sunburn, andconjunctivitis.

[0101] The term “treating”, as used herein, refers to reversing,alleviating, inhibiting the progress of, or preventing the disorder orcondition to which such term applies, or one or more symptoms of suchdisorder or condition. The term “treatment”, as used herein, refers tothe act of treating, as “treating” is defined immediately above.

[0102] This invention also encompasses pharmaceutical compositions forthe treatment of a condition selected from the group consisting ofarthritis, psoriatic arthritis, Reiter's syndrome, gout, traumaticarthritis, rubella arthritis and acute synovitis, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, Alzheimer's disease, stroke,neurotrauma, asthma, adult respiratory distress syndrome, cerebralmalaria, chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, bone resorption disease, osteoporosis, restenosis, cardiacand renal reperfusion injury, thrombosis, glomerularonephritis,diabetes, graft vs. host reaction, allograft rejection, inflammatorybowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis,muscle degeneration, eczema, contact dermatitis, psoriasis, sunburn, orconjunctivitis shock in a mammal, including a human, comprising anamount of a compound of formula I effective in such treatment and apharmaceutically acceptable carrier.

[0103] This invention also encompasses pharmaceutical compositions forthe treatment of a condition which can be treated by the inhibition ofMAP kinase in a mammal, including a human, comprising an amount of acompound of claim 1 effective in such treatment and a pharmaceuticallyacceptable carrier.

[0104] This invention also encompasses pharmaceutical compositions forthe treatment of a condition which can be treated by the inhibition ofp38 kinase in a mammal, including a human, comprising an amount of acompound of claim 1 effective in such treatment and a pharmaceuticallyacceptable carrier.

[0105] This invention also encompasses pharmaceutical compositionscontaining prodrugs of compounds of the formula I. Compounds of formulaI having free amino, amido, hydroxy or carboxylic groups can beconverted into prodrugs. Prodrugs include compounds wherein an aminoacid residue, or a polypeptide chain of two or more (e.g., two, three orfour) amino acid residues which are covalently joined through peptidebonds to free amino, hydroxy or carboxylic acid groups of compounds offormula I. The amino acid residues include the 20 naturally occurringamino acids commonly designated by three letter symbols and alsoinclude, 4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Prodrugs also include compounds wherein carbonates, carbamates, amidesand alkyl esters which are covalently bonded to the above substituentsof formula I through the carbonyl carbon prodrug sidechain.

[0106] The invention also encompasses sustained release compositions.

[0107] One of ordinary skill in the art will appreciate that thecompounds of the invention are useful in treating a diverse array ofdiseases. One of ordinary skill in the art will also appreciate thatwhen using the compounds of the invention in the treatment of a specificdisease that the compounds of the invention may be combined with variousexisting therapeutic agents used for that disease.

[0108] For the treatment of rheumatoid arthritis, the compounds of theinvention may be combined with agents such as TNF-α inhibitors such asanti-TNF monoclonal antibodies (such as Remicade, CDP-870 and D₂E₇) andTNF receptor immunoglobulin molecules (such as Enbrel®), IL-1inhibitors, receptor antagonists or soluble IL-1ra (e.g. Kineret or ICEinhibitors), COX-2 inhibitors (such as celecoxib, rofecoxib, valdecoxiband etoricoxib), metalloprotease inhibitors (preferably MMP-13 selectiveinhibitors), p2×7 inhibitors, α2δ inhibitors, low dose methotrexate,leflunomide, hydroxychloroquine, d-penicillamine, auranofin orparenteral or oral gold.

[0109] The compounds of the invention can also be used in combinationwith existing therapeutic agents for the treatment of osteoarthritis.Suitable agents to be used in combination include standard non-steroidalanti-inflammatory agents (hereinafter NSAID's) such as piroxicam,diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen,ketoprofen and ibuprofen, fenamates such as mefenamic acid,indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone,salicylates such as aspirin, COX-2 inhibitors such as celecoxib,valdecoxib, rofecoxib and etoricoxib, analgesics and intraarticulartherapies such as corticosteroids and hyaluronic acids such as hyalganand synvisc.

[0110] The compounds of the present invention may also be used incombination with anticancer agents such as endostatin and angiostatin orcytotoxic drugs such as adriamycin, daunomycin, cis-platinum, etoposide,taxol, taxotere and alkaloids, such as vincristine, farnesyl transferaseinhibitors, VegF inhibitors, and antimetabolites such as methotrexate.

[0111] The compounds of the invention may also be used in combinationwith antiviral agents such as Viracept, AZT, aciclovir and famciclovir,and antisepsis compounds such as Valant.

[0112] The compounds of the present invention may also be used incombination with cardiovascular agents such as calcium channel blockers,lipid lowering agents such as statins, fibrates, beta-blockers, Aceinhibitors, Angiotensin-2 receptor antagonists and platelet aggregationinhibitors.

[0113] The compounds of the present invention may also be used incombination with CNS agents such as antidepressants (such assertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa, Requip,Mirapex, MAOB inhibitors such as selegine and rasagiline, comPinhibitors such as Tasmar, A-2 inhibitors, dopamine reuptake inhibitors,NMDA antagonists, Nicotine agonists, Dopamine agonists and inhibitors ofneuronal nitric oxide synthase), and anti-Alzheimer's drugs such asdonepezil, tacrine, α2δ inhibitors, COX-2 inhibitors, propentofylline ormetryfonate.

[0114] The compounds of the present invention may also be used incombination with osteoporosis agents such as roloxifene, droloxifene,lasofoxifene or fosomax and immunosuppressant agents such as FK-506 andrapamycin.

DETAILED DESCRIPTION OF THE INVENTION

[0115] Compounds of the formula I may be prepared according to thefollowing reaction schemes and discussion. Unless otherwise indicated s,R¹ and R² and structural formula I (and Ia and Ib) in the reactionschemes and discussion that follow are as defined above.

[0116] Scheme 1 refers to the preparation of compounds of the formula Iin two steps from compounds of formula III. Referring to Scheme 1compounds of the formula III, wherein L is a suitable leaving group suchas fluoro, bromo, chloro or mesyl (MeSO₂), preferably bromo or chloro,are converted to the corresponding compound of formula II by reactionwith hydrazine to form a hydrazino-pyridine, followed by reaction withan acylating reagent. The reaction of a compound of formula III withhydrazine is conducted in a polar solvent such as pyridine, ethanol ortert-butanol, or in neat hydrazine, preferably in neat hydrazine. Thehydrazine reaction is conducted at a temperature between about 40° C. toabout 80° C., preferably about 70° C. for about 10 minutes to about 60minutes, preferably about 15 minutes. Acylation of the resultinghydrazino-pyridine to give compounds of the formula II is conducted withan acid chloride in the presence of a base such as triethylamine in asolvent such as dichloromethane, tetrahydrofuran, N,N-dimethylformamide,preferably dichloromethane, for a time period between about 10 minutesto about 120 minutes, preferably about 30 minutes, at a temperature ofabout 0° C. to about 22° C., preferably at about 0° C. Alternatively,the hydrazino-pyridine can be acylated with a carboxylic acid to givecompounds of the formula II using amide coupling agents in a manner wellknown to one skilled in the art.

[0117] The compound of formula II can be converted to a compound offormula I using a suitable dehydrating agent or under conditions thatpromote cyclo-dehydration. Suitable dehydrating agents for theconversion of compounds of formula II to compounds of formula I includephosphorous oxychloride and dichlorotriphenylphosphorane, preferablyphosphorous oxychloride. Reactions using phosphorous oxychloride areconducted in neat phosphorous oxychloride at a temperature between about60° C. to about 110° C., for a time period between about 2 hours toabout 16 hours. Reactions using dichlorotriphenylphosphorane areconducted in the presence of a base, such as triethylamine, in a polarsolvent such as acetonitrile, at temperatures of about 60° C. and refluxfor a time period from about 1 hour and about 8 hours.

[0118] Compounds of the formula III can be made according to the methodsof Scheme 2.

[0119] Scheme 2 refers to the preparation of compounds of the formulaIII, which are intermediates useful in the preparation of compounds ofthe formula I, in Scheme 1. Referring to Scheme 2, a compound of theformula III, can be prepared from a compound of formula IV, by heatingwith formamide. The aforesaid reaction can be run at a temperature fromabout 100° C. to about 160° C. for a period from about 1 hour to about12 hours, preferably at about 160° C. for about 3 hours.

[0120] The compound of formula IV is prepared from a compound of formulaV by reaction with sodium methoxide, or sodium ethoxide, or sodiumtert-butoxide, preferably sodium methoxide, in an alcohol solvent, suchas methanol, ethanol, isopropanol, preferably methanol, at a temperatureof 0° C. to 30° C., preferably at 22° C., for a period of time from 15minutes to about 3 hours, preferably 30 minutes. The aforesaid reactionis followed by an aqueous acidic work-up.

[0121] The compound of formula V is prepared from a compound of formulaVI by reaction with Br₂ in a polar solvent. Suitable solvents includeacetic acid, chloroform or methylene chloride, preferably acetic acid.The aforesaid reaction is conducted at a temperature of about 0° C. toabout 30° C. preferably at about 22° C. (room temperature) for a periodfrom about 10 minutes to about 4 hours, preferably about 30 minutes.

[0122] The compounds of formula IV can also be prepared according to themethods of Scheme 4. The compounds of formula VI are prepared accordingto the methods of Scheme 5. Additional routes for the synthesis ofcompounds related to formula VI are described in the literature: Davies,I. W.; Marcoux, J. F.; Corley, E. G.; Journet, M.; Cai, D. W.; Palucki,M.; Wu, J.; Larsen, R. D.; Rossen, K.; Pye, P. J.; DiMichele, L.;Dormer, P.; Reider, P. J.; J. Org. Chem., Vol. 65, pp. 8415-8420 (2000).

[0123] Scheme 3 refers to an alternate preparation of compounds offormula III, which are intermediates in Scheme 1. Referring to Scheme 3,compounds of the formula III, can be prepared from compounds of formulaVIII by reaction with an isocyanide of formula

[0124] in the presence of a base. Suitable bases include potassiumcarbonate, triethylamine, 2,6-lutidine and piperazine, preferably2,6-lutidine. Suitable solvents include polar solvents such astetrahydrofuran, acetonitrile or N,N-dimethylformamide, preferably inacetonitrile or tetrahydrofuran. The aforesaid reaction may be run at atemperature between about 22° C. and about 70° C., preferably at about22° C. for a period from about 2 hours to about 4 hours, followed byabout 6 hours to about 10 hours at a temperature of about 70° C.

[0125] Compounds of formula VIII are known in the literature (when L ischloro see: Corey, E. J.; Loh, T. P.; Achyutha Rao, S.; Daley, D. C.;Sarshar, S., J. Org. Chem., 1993, 58, 5600-5602) or can be prepared in amanner well known to one skilled in the art.

[0126] Compounds of the formula

[0127] may be prepared by reacting a compound of the formula

[0128] with a dehydrating agent such as POCl₃, and a weak hindered basesuch as 2,6 lutidine or 2,4,6-trimethylpyridine. Preferably the reactionis performed in the presence of a solvent such as tetrahydrofuran,dimethyl ether or methylene chloride. The aforesaid reaction may be runat a temperature between about −20° C. and about 50° C., preferably atabout 0° C. to about room temperature for a period from about 2 hours toabout 48 hours, preferably about 24 hours.

[0129] Scheme 4 refers to an alternate preparation of compounds offormula IV, which are intermediates in Scheme 2, useful in thepreparation of compounds of formula I.

[0130] Compounds of formula IV can be prepared from compounds of formulaIX by reaction with a suitably substituted Grignard reagent of theformula (R¹)_(s)-phenyl-M, wherein M is an activation group such asmagnesium bromide or chloride (see for example: Jackson, W. R.; Jacobs,H. A.; Jayatilake, G. S.; Matthews, B. R.; Watson, K. G., Aust. J.Chem., 1990, 43, 2045-2062). Reagents of the formula (R¹)_(s)-phenyl-Mare commercially available or may be prepared by one skilled in the art.

[0131] The preparation and conversion of compounds of formula X intotrimethylsilyl cyanohydrins of formula IX can be performed by methodsknown to those skilled in the art such as for example Pirrung, M.;Shuey, S. W.; J. Org. Chem., 1994, 59, 3890-3897.

[0132] Scheme 5 refers to the preparation of compounds of the formulaVI, which are intermediates for the preparation of compounds of formulaIII in Scheme 2. Referring to Scheme 5, a compound of the formula VI isprepared from a compound of formula XI by reaction with a Grignardreagent of the formula (R¹)_(s)-phenyl-M, wherein M is an activatinggroup such as magnesium bromide or magnesium chloride in a solvent.Suitable solvents include tetrahydrofuran, dioxane, dimethylethyl etheror diethyl ether, preferably tetrahydrofuran. The aforesaid reaction isconducted at a temperature of about −78° C. to 0° C. for a period fromabout 10 minutes to about 24 hours preferably about 2 hours. Reagents ofthe formula (R¹)_(s)-phenyl-M are commercially available or may beprepared by one skilled in the art.

[0133] A compound of formula XI is prepared from a compound of formulaXII by reaction with a hydroxylamine of the formula

[0134] wherein P² and P³ are independently (C₁-C₆)alkyl, preferablymethyl, and an activating agent. Suitable activating agents includecarbonyldiimidazole or oxalyl chloride, preferably carbonyldiimidazole.Suitable solvents include methylene chloride or dichloroethane.

[0135] Compounds of the formula XII are prepared from compounds offormula XIV by acid hydrolysis, such as by reaction with sulfuricacid/water (preferably 1:1) at a temperature of about 100° C. to about120° C., preferably about 110° C. for a period from about 1 hour toabout 6 hours, preferably about 4 hours. Alternatively, a compound ofthe formula XII is prepared by base hydrolysis, such as by reaction withlithium hydroxide in water at a temperature of about 23° C. to about100° C., preferably at a temperature of about 80° C. for a period ofabout 4 to 10 hours.

[0136] Scheme 6 refers to an alternate preparation of compounds offormula 1. Referring to Scheme 6, compounds of the formula I can beprepared from compounds of the formula XV by reaction with a boronicester of the formula

[0137] a transition metal catalyst, and a base. Suitable catalystsinclude copper or palladium (such as palladium acetate (Pd(OAc)₂),tetrakis(triphenylphosphine) palladium (0) or Pd(dppf)Cl₂), preferablytetrakis(triphenylphosphine) palladium (0). Suitable bases includetertiary amine bases, such as triethylamine or pyridine, Na₂CO₃, sodiumethoxide, and K₃PO₄, preferably triethylamine. Suitable solvents includealcohols, such as methanol, ethanol and butanol, methylene chloride,dimethyl sulfoxide (DMSO) or tetrahydrofuran (THF), preferably ethanol.The aforesaid reaction is typically performed under an atmosphere ofnitrogen gas at a temperature of about 10° C. to 85° C., preferablyabout 70° C. for about 6 to 72 hours. Palladiumcatalyzed boronic acidcouplings are described in Miyaura, N., Yanagi, T., Suzuki, A., Syn.Comm., 1981, 11, 7, p. 513.

[0138] The compound of formula XV is prepared from a compound of formulaXVII by reaction with a suitable bromination reagent such as phenyltrimethylammonium tribromide, N-bromosuccinimide, pyridinium bromide,perbromide, Br₂ or Br₂—Ph₃P, preferably N-bromosuccinimide. Thebromination may be carried out in a reaction inert solvent such asN,N-dimethylformamide, diethyl ether or tetrahydrofuran, preferablydimethyl formamide. The aforesaid reaction is conducted at a temperatureof about −78° C. to about 40° C. preferably about −78° C. to about 0° C.for a time period between about 1 hour to about 16 hours. Preferably,the reaction is conducted in the presence of a base such as lithiumbis(trimethylsilyl(amide)).

[0139] The compound of formula XVII is prepared from a compound of theformula XVIII by reaction with tosylmethylisocyanide in the presence ofa base in a solvent. Suitable bases include alkali metal carbonates orhydroxide bases, preferably potassium carbonate. Suitable solvents forthe aforesaid reaction include hexane, methylene chloride, alcohols,N,N-dimethylformamide (DMF), N,N-dimethylacetamide orN-methylpyrrolidinone (NMP) preferably methanol. The aforesaid reactionmay be run at a temperature between about 30° C. and 180° C., preferablyabout 65° C., for about 30 minutes to 24 hours, preferably about 2hours.

[0140] Alternatively, a compound of the formula I can be prepared fromaldehydes of formula XVIII as described previously in Scheme 3 for theconversion of compounds of formula VIII to compounds of formula III.

[0141] Compounds of formula XVIII are prepared from compounds of formulaXIX, wherein L′ is bromo or iodo, by a formylation reaction. Suitableconditions for formylation include metal halogen exchange withisopropylmagnesium chloride in a solvent such as tetrahydrofuran at atemperature of about 0° C., for a period of time of about 30 minutes,followed by the addition of N,N-dimethylformamide at a temperature ofabout 0° C., followed by a period of time of about 2.5 hours at atemperature of about 50° C.

[0142] Compounds of formula XIX are prepared as described in theliterature (Moran, D. B.; Morton, G. O.; Albright, J. D., J. Heterocycl.Chem., Vol. 23, pp. 1071-1077 (1986)) or from compounds of formula XX asdescribed in Scheme 1 for the conversion of compounds of formula III tocompounds of formula I. Compounds of formula XX are commerciallyavailable.

[0143] The compounds of the formula I which are basic in nature arecapable of forming a wide variety of different salts with variousinorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to animals, it is oftendesirable in practice to initially isolate a compound of the formula Ifrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent, and subsequently convert the freebase to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isobtained.

[0144] The acids which are used to prepare the pharmaceuticallyacceptable acid addition salts of the base compounds of this inventionare those which form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, such as hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate or bisulfate, phosphate oracid phosphate, acetate, lactate, citrate or acid citrate, tartrate orbitartrate, succinate, maleate, fumarate, gluconate, saccharate,benzoate, methanesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.

[0145] Those compounds of the formula I which are also acidic in nature,are capable of forming base salts with various pharmacologicallyacceptable cations. Examples of such salts include the alkali metal oralkaline-earth metal salts and particularly, the sodium and potassiumsalts. These salts are all prepared by conventional techniques. Thechemical bases which are used as reagents to prepare thepharmaceutically acceptable base salts of this invention are those whichform non-toxic base salts with the herein described acidic compounds offormula I. These non-toxic base salts include those derived from suchpharmacologically acceptable cations as sodium, potassium, calcium andmagnesium, etc. These salts can easily be prepared by treating thecorresponding acidic compounds with an aqueous solution containing thedesired pharmacologically acceptable cations, and then evaporating theresulting solution to dryness, preferably under reduced pressure.Alternatively, they may also be prepared by mixing lower alkanolicsolutions of the acidic compounds and the desired alkali metal alkoxidetogether, and then evaporating the resulting solution to dryness in thesame manner as before. In either case, stoichiometric quantities ofreagents are preferably employed in order to ensure completeness ofreaction and maximum product yields.

[0146] The activity of the compounds of the invention for the variousdisorders described above can be determined according to one or more ofthe following assays. All of the compounds of the invention, that weretested, had an IC₅₀ of less than 10 μM in the TNFαand MAPKAP in vitroassays and an ED₅₀ of less than 50 mg/kg in the in vivo TNFα assay.

[0147] The compounds of the present invention also possess differentialactivity (i.e. are selective for) for one or more p38 kinases (i.e. α,β, γ, and δ) or other MAP kinases. Certain compounds are selective forp38α over p38β, γ, and δ, other compounds are selective for p38β, overp38α, γ, and δ, other compounds are selective for p38 α and β over p38 γand δ. Selectivity is measured in standard assays as a IC₅₀ ratio ofinhibition in each assay.

[0148] Inhibition of TNF-ALPHA Production by Human LPS-Treated Monocytes

[0149] Mononuclear cells are isolated from heparinized blood (1.5 ml of1000 units/ml heparin for injection, Elkins-Sinn, Inc. added to each 50ml sample) using Accuspin System-Histopaque-1077 tubes (Sigma A-7054).Thirty-five milliliters of whole blood are added to each tube and thetubes are centrifuged at 2100 rpm for 20 minutes in a Beckman GS-6KRcentrifuge with the brake off at room temperature. The mononuclear cellswhich collect at the interface are removed, diluted with Macrophageserum free medium (Gibco-BRL) (Medium) to achieve a final volume of 50ml, and collected by centrifugation for 10 minutes. The supernatant isdiscarded and the cell pellet is washed 2 times with 50 ml of Medium. Asample of the suspended cells is taken before the second wash forcounting. Based on this count, the washed cells are diluted with Mediumcontaining 1% FBS to a final concentration of 2.7×10⁶ cells/ml and 75 μlof the cell suspension is added to each well of a 96 well plate.

[0150] Compound Preparation

[0151] Compounds are routinely tested at final concentrations from 2 μMto 0.016 μM, but may be tested at other concentrations, depending onactivity. Test agents are diluted with DMSO to a final concentration of2 mM. From this stock solution, compounds are first diluted 1:25 (5 μlof 2 mM stock +120 μl Medium containing 400 ng/ml LPS and 1% FBS then 40μl of this dilution is diluted with 360 μl of Medium with LPS. Serialdilutions (1/5) are performed by transferring 20 μl of this dilution to80 μl of Medium containing both LPS and 0.4% DMSO, resulting insolutions containing 8 μM, 1.6 μM, 0.32 μM and 0.064 μM of test agent.

[0152] Assay

[0153] The assay is initiated by adding 25 μl of the diluted compoundsto the mononuclear cell suspension and incubating the cells at 37 C and5% CO₂ for 4 hours.

[0154] The 96-well plates are then centrifuged for 10 minutes at 2000rpm at 4° C. in a Beckman GS-6KR centrifuge to remove cells and celldebris. A 90 μl aliquot of each supernatant is removed and transferredto a 96 well round bottom plate, and this plate is centrifuged a secondtime to insure that all cell debris is removed. 80 μl of the supernatantis removed and transferred to a new round bottom plate.

[0155] Supernatants are analyzed for TNF-α content using R&D ELISA. 25μl of each sample is added to an ELISA well containing 25 μl of assaydiluent RD1 F and 75 μl of assay diluent RD5. The assay is run followingkit directions except 100 μl of conjugate and substrate solutions areused.

Interpretation

[0156] The amount of TNF-a immunoreactivity in the samples is calculatedas follows:

% Control=(X−B)/(TOT−B)×100

[0157] where X=OD₄₅₀ nm of the test compound well

[0158] B=OD₄₅₀ of Reagent Blank wells on the ELISA

[0159] Total=OD₄₅₀ of cells that were treated with 0.1% DMSO only.

MAPKAP Kinase-2 Assay

[0160] Monocyte Preparation

[0161] Mononuclear cells are collected from heparinized human blood asdetailed above. The washed cells are seeded into 6-well cluster platesat a density of 1×10⁷ cells/well (in 2 ml of Medium). The plates areincubated at 37° C. in a 5% CO₂ environment for 2 hours to allowadherence of the monocytes, after which time media supernatantscontaining non-adherent cells are removed by aspiration and 2 ml offresh medium are added to each well. Plates are incubated overnight at37° C. in a 5% CO₂ environment.

[0162] Cell Activation

[0163] Media are removed by aspiration. The attached cells are rinsedtwice with fresh Medium, then 2 ml of D-MEM medium containing 10% heatinactivated FBS are added to each well. Test compounds are prepared as30 mM stock solutions in DMSO and diluted to 1250, 250, 50, 10, 2, and0.4 μM in D-MEM containing 1% DMSO and 10% FBS. To individual wells ofthe monocyte cultures, 20 μl of these test agent dilutions are addedresulting in final test agent concentrations of 12.5, 2.5, 0.5, 0.1,0.02 and 0.004 μM. After a 10 minute preincubation period, 20 μl of a 10μg/ml LPS solution are added to each well and the plates are incubatedat 37° C. for 30 minutes. Media subsequently are removed by aspiration,the attached monocytes are rinsed twice with phosphate buffered saline,then 1 ml of phosphate buffered saline containing 1% Triton X-100 (LysisBuffer; also containing 1 Complete tablet [Boehringer #1697498] per 10ml of buffer) is added to each well. The plates are incubated on ice for10 minutes, after which the lysates are harvested and transferred tocentrifugation tubes. After all samples are harvested, they areclarified by centrifugation (45,000 rpm for 20 min) and the supernatantsrecovered.

[0164] MAPKAP Kinase-2 Immunoprecipitation

[0165] 5 μl of anti-MAPKAP kinase-2 antiserum (Upstate Biotechnology#06-534) is added to a microcentrifuge tube (1 tube for each of theabove cell lysates) containing 1 ml of a 5% suspension of ProteinG-Sepharose (Sigma #P3296) in PBS. These mixtures are incubated for 1hour at 4° C. (with rocking) after which the beads, containing boundIgG, are recovered by centrifugation and washed twice with 1 ml of 50 mMTris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM orthovanadate, 0.1%2-mercaptoethanol, 1% Triton X-100, 5 mM sodium pyrophosphate, 10 mMsodium β-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 μg/mlleupeptin, 1 μg/ml pepstatin, and 50 mM sodium fluoride (Buffer A) byrepeated centrifugation. An individual monocyte cell extract (preparedabove) is then transferred to each tube containing a pellet ofIgG-coated Protein G-Sepharose, and these mixtures are incubated for 2hours at 4° C. (with rocking). The beads subsequently are harvested bycentrifugation, and the resulting bead pellets are washed once with 0.5ml of Buffer A containing 0.5 M NaCl, once with 0.5 ml of Buffer A, andonce with 0.1 ml of a buffer composed of 20 mM MOPS, pH 7.2, 25 mMsodium β-glycerophosphate 5 mM EGTA, 1 mM orthovanadate, and 1 mMdithiothreitol (Buffer B).

[0166] MAPKAP Kinase-2 Activity Assessment

[0167] A kinase reaction mixture stock is prepared as follows: 2.2 μl of10 mCi/ml γ[³²P]ATP, 88 μl of 1.3 μg/ml solution of MAPKAP Kinase-2substrate peptide (Upstate Biotechnology #12-240), 11 μl of 10 mM ATP,8.8 μl of 1 M MgCl₂, and 770 μl of Buffer B. To each of the immunecomplex-Protein G-pellets, 40 μl of the kinase reaction mixture areadded and the tubes are incubated for 30 minutes at 30° C. The tubesthen are clarified by centrifugation and 25 μl of each supernatant isspotted onto a P81 filter paper disk (Whatman #3698-023). After allowingall fluid to soak into the filter, each disk is placed into anindividual well of 6-well cluster plates and the filters are washedsequentially with 2 ml of 0.75% phosphoric acid (3 washes/15 minuteseach) and once with acetone (10 minutes). The filters then are air driedand transferred to liquid scintillation vials containing 5 ml ofscintillation fluid. Radioactivity is determined in a liquidscintillation counter. The amount of radioactivity bound to the filterat each test agent concentration is expressed as a percentage of thatobserved from cells stimulated with LPS in the absence of a test agent.

[0168] In Vivo Inhibition of TNFα

[0169] Rats were weighed and dosed with vehicle (0.5% methylcellulose,Sigma) or drug. One hour later, animals were injected i.p. with LPS (50ug/rat, Sigma L-4130). Ninety minutes later, animals were sacrificed byasphyxiation with CO₂ and bled by cardiac puncture. Blood was collectedin Vaccutainer tubes and spun for 20 minutes at 3000 rpm. Serum wasassayed for TNFα levels using an ELISA (R&D Systems).

[0170] This invention also encompasses pharmaceutical compositionscontaining and methods of treating or preventing comprisingadministering prodrugs of compounds of the formula I. Compounds offormula I having free amino, amido, hydroxy or carboxylic groups can beconverted into prodrugs. Prodrugs include compounds wherein an aminoacid residue, or a polypeptide chain of two or more (e.g., two, three orfour) amino acid residues which are covalently joined through peptidebonds to free amino, hydroxy or carboxylic acid groups of compounds offormula I. The amino acid residues include the 20 naturally occurringamino acids commonly designated by three letter symbols and alsoinclude, 4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gammaminobutyric acid,citrulline homocysteine, homoserine, omithine and methionine sulfone.Prodrugs also include compounds wherein carbonates, carbamates, amidesand alkyl esters which are covalently bonded to the above substituentsof formula I through the carbonyl carbon prodrug sidechain.

[0171] The compositions of the present invention may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers. Thus, the active compounds of the invention may be formulatedfor oral, buccal, intranasal, parenteral (e.g., intravenous,intramuscular or subcutaneous) or rectal administration or in a formsuitable for administration by inhalation or insufflation.

[0172] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium phosphate); lubricants (e.g., magnesium stearate,talc or silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, methyl cellulose or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters or ethyl alcohol); and preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

[0173] For buccal administration, the composition may take the form oftablets or lozenges formulated in conventional manner.

[0174] The compounds of formula I can also be formulated for sustaineddelivery according to methods well known to those of ordinary skill inthe art. Examples of such formulations can be found in U.S. Pat. Nos.3,538,214, 4,060,598, 4,173,626, 3,119,742, and 3,492,397, which areherein incorporated by reference in their entirety.

[0175] The active compounds of the invention may be formulated forparenteral administration by injection, including using conventionalcatheterization techniques or infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0176] The active compounds of the invention may also be formulated inrectal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

[0177] For intranasal administration or administration by inhalation,the active compounds of the invention are conveniently delivered in theform of a solution or suspension from a pump spray container that issqueezed or pumped by the patient or as an aerosol spray presentationfrom a pressurized container or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. The pressurized containeror nebulizer may contain a solution or suspension of the activecompound. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated containing a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

[0178] A proposed dose of the active compounds of the invention fororal, parenteral or buccal administration to the average adult human forthe treatment of the conditions referred to above (e.g., inflammation)is 0.1 to 200 mg of the active ingredient per unit dose which could beadministered, for example, 1 to 4 times per day.

[0179] Aerosol formulations for treatment of the conditions referred toabove (e.g., adult respiratory distress syndrome) in the average adulthuman are preferably arranged so that each metered dose or “puff” ofaerosol contains 20 μg to 1000 μg of the compound of the invention. Theoverall daily dose with an aerosol will be within the range 100 μg to 10mg. Administration may be several times daily, for example 2, 3, 4 or 8times, giving for example, 1, 2 or 3 doses each time.

[0180] Aerosol combination formulations for treatment of the conditionsreferred to above in the average adult human are preferably arranged sothat each metered dose or “puff” of aerosol contains from about 0.01 mgto about 100 mg of the active compound of this invention, preferablyfrom about 1 mg to about 10 mg of such compound. Administration may beseveral times daily, for example 2, 3, 4 or 8 times, giving for example,1, 2 or 3 doses each time.

[0181] Aerosol formulations for treatment of the conditions referred toabove in the average adult human are preferably arranged so that eachmetered dose or “puff” of aerosol contains from about 0.01 mg to about2000 mg of an MAP kinase inhibitor, preferably from about 1 mg to about200 mg of p38 kinase inhibitor. Administration may be several timesdaily, for example 2, 3, 4 or 8 times, giving for example, 1, 2 or 3doses each time.

[0182] The following Examples illustrate the preparation of thecompounds of the present invention. Melting points are uncorrected. NMRdata are reported in parts per million (δ) and are referenced to thedeuterium lock signal from the sample solvent (deuteriochloroform unlessotherwise specified). Mass Spectral data were obtained using a MicromassZMD APCI Mass Spectrometer equipped with a Gilson gradient highperformance liquid chromatograph. The following solvents and gradientswere used for the analysis. Solvent A; 98% water/2% acetonirile/0.01%formic acid and solvent B; acetonitrile containing 0.005% formic acid.Typically, a gradient was run over a period of about 4 minutes startingat 95% solvent A and ending with 100% solvent B. The mass spectrum ofthe major eluting component was then obtained in positive or negativeion mode scanning a molecular weight range from 165 amu to 1100 amu.Specific rotations were measured at room temperature using the sodium Dline (589 nm). Commercial reagents were utilized without furtherpurification. THF refers to tetrahydrofuran. DMF refers toN,N-dimethylformamide. Chromatography refers to column chromatographyperformed using 32-63 mm silica gel and executed under nitrogen pressure(flash chromatography) conditions. Room or ambient temperature refers to20-25° C. All non-aqueous reactions were run under a nitrogen atmospherefor convenience and to maximize yields. Concentration at reducedpressure means that a rotary evaporator was used.

[0183] One of ordinary skill in the art will appreciate that in somecases, protecting groups may be required during preparation. After thetarget molecule is prepared, the protecting group can be removed bymethods well known to those of ordinary skill in the art, such asdescribed in Greene and Wuts, Protective Groups in Organic Synthesis,(2^(nd) Ed., John Wiley & Sons, 1991).

Preparation 1 5-BROMO-PYRIDIN-2-YL-HYDRAZINE

[0184]

[0185] A 12 L three-necked round-bottomed flask equipped with amechanical stirrer and a condenser, connected on top with a nitrogenbubbler and a thermometer, was charged with 2,5-dibromopyridine (442 g,1.87 moles), hydrazine hydrate (55% wt., 1057 ml, 18.7 moles),poly(ethylene glycol) (average Mn about 300, 1.87 L), 2-butanol (373 ml)and water (1.87 L). The mixture was heated at reflux for 29 hours. Theheating source was removed and the mixture was stirred for an additional20 hours. To the resulting slurry, cold water (2.2 L) was added. Theslurry was stirred for an additional 30 minutes and filtered. The cakewas washed with cold water (3×200 ml) and dried in a vacuum-oven (40°C.) for 48 hours. The title compound was obtained as off-white flakes(305 g, yield 87%).

[0186] GCMS(m/z): 187 (M+). H¹ NMR (400 MHz, CDCl₃): δ 8.14 (d, J=2.0Hz, 1H), 7.55 (dd, J=8.7/2.0 Hz, 1H), 6.66 (d, J=8.7 Hz, 1H), 5.89 (brs,1H), 3.65 (brs, 2H).

Preparation 2 6-BROMO-3-ISOPROPYL-[1,2,4]TRIAZOLO(4.3-A)PYRIDINEHYDROCHLORIDE

[0187]

[0188] A 500 ml three-necked round-bottomed flask equipped with amechanical stirrer and a condenser, connected on top to a nitrogenbubbler and a thermometer, was charged with5-bromo-pyridin-2-yl-hydrazine (43.4 g, 0.231 moles) and isobutyrylchloride (218 ml, 2.08 moles). The mixture was gently refluxed for 3hours. The heating source was then replaced with an ice-water bath andthe slurry cooled to room temperature. Hexane (220 ml) was added and theslurry stirred at room temperature for 15 minutes and filtered. The cakewas washed with hexane (3×70 ml) and then dried in a vacuum-oven (35°C.) for 48 hours. The title compound was obtained as an off-white powder(58.96 g, yield 92.3%).

[0189] Preparation 3

6-BROMO-3-ISOPROPYL-[1,2,4]TRIAZOLO(4,3-A)PYRIDINE

[0190]

[0191] A 5 L three-necked round-bottomed flask, equipped with amechanical stirrer and a thermometer, was charged with6-bromo-3-isopropyl-[1,2,4]triazolo(4,3-a)pyridine hydrochloride (587.0g, 2.12 moles), water (1.2 L) and dichloromethane (1.8 L). The biphasicmixture was cooled to 5 to 10° C. using an ice-water bath. Sodiumhydroxide (1N aqueous solution) (2.15 L) was added over a period of 10minutes. The mixture was stirred in the bath for 15 minutes. The organiclayer was then isolated and the aqueous layer extracted withdichloromethane (600 mL). The combined organic extracts are washed with1:1 brine-water (2 L) and dried (MgSO₄). Most of dichloromethane wasremoved by rotary evaporation. Ethyl acetate (800 ml) was then added.After removing about 400 ml of solvents, hexane (3.2 L) was added. Theslurry was stirred in an ice-water bath for 2 hours and then filtered.The cake was washed with 9:1 hexane-ethyl acetate (3×150 ml) and driedin a vacuum-oven (30-35° C.) for 18 hours. The title compound (471.6 g,yield 92.5%), was obtained as a tan sandy powder.

[0192] H¹ NMR (400 MHz, CDCl₃): δ 8.06 (s, 1H), 7.64 (d, J=9.5 Hz, 1H),7.24 (d, J=9.5 Hz, 1H), 3.33 (m, J=7.0 Hz, 1H), 1.52 (d, J=7.0 Hz, 6H).

Preparation 43-ISOPROPYL-[1,2,4]TRIAZOLO(4,3-A)-6-PYRIDINECARBOXALDEHYDE

[0193]

[0194] A 12 L three-necked round-bottomed flask, equipped with amechanical stirrer, an addition funnel and a thermometer, was chargedwith 6-bromo-3-isopropyl-[1,2,4]triazolo(4,3-a)pyridine (200.0 g, 0.833moles) and tetrahydrofuran (J. T. Baker, low water 2.0 L). The solutionwas cooled to −8° C. using an acetone/dry ice bath. A solution ofisopropylmagnesium chloride in tetrahydrofuran (2.0M, 500 ml, 1.0 mole)L) was added via the addition funnel over a period of 55 minutes. Theresulting brownish slurry was stirred between −4 to 0° C. for 30minutes. Dimethylformamide (Aldrich, anhydrous, 155 ml, 2.0 moles) wasadded via an addition funnel over a period of 5 minutes. The coolingbath was replaced with a heating mantle and the addition funnel wasreplaced with a condenser. The slurry was heated to 55° C. and stirredat this temperature for 2 hours. The reaction mixture was cooled to 15°C. and dichloromethane (3 L) was added. The slurry was slowly pouredinto a stirred and ice-water cooled (15° C.) 10% by weight aqueoussolution of citric acid (3 kg) over a period of 5 minutes. The biphasicmixture was stirred at 17 to 20° C. for 30 minutes. The organic layerwas then isolated and the aqueous layer extracted with dichloromethane(5×1 L). The combined organic extracts were washed with 1:1 v/vbrine-water (2 L), dried (MgSO₄) and concentrated. To the brownishresidual solid was added ethyl acetate (800 ml). The slurry was stirredat room temperature for 10 minutes at which time hexane (800 ml) wasadded. The slurry was stirred at room temperature for 2 more hours andfiltered. The cake was washed with 1:1 v/v hexane-ethyl acetate (3×150ml) and dried in a vacuum-oven (30-35° C.) for 18 hours. The titlecompound was obtained as a yellowish sandy powder (126.6 g, yield 80%).

[0195] GCMS(m/z): 189 (M+). H¹ NMR (400 MHz, CDCl₃): δ 10.00 (s, 1H),8.49 (s, 1H), 7.79 (d, J=9.5 Hz, 1H), 7.68 (d, J=9.5 Hz, 1H), 3.47 (m,J=7.0 Hz, 1H), 1.56 (d, J=7.0 Hz, 6H).

Preparation 5 P-TOLUENESULFINIC ACID

[0196]

[0197] A 5 L three-necked round-bottomed flask, equipped with amechanical stirrer and a thermometer, was charged with p-toluenesulfinicacid, sodium salt hydrate (Aldrich, CH₃C₆H₄SO₂Na.xH₂O, 392.0 g), tapwater (2 L) and methyl t-butyl ether (2 L). The mixture was stirred atroom temperature for 10 minutes at which time hydrochloric acid (37% wt.in water, 142 ml, 1.2 moles) was added over a period of 5 minutes. Thebiphasic mixture was stirred at room temperature for 30 minutes. Theorganic layer was then isolated and the aqueous layer extracted withmethyl t-butyl ether (500 mL). The combined organic extracts wereconcentrated to a residual white semi-solid, which was diluted withtoluene (700 ml). Most of solvents were removed and hexane (1.8 L) wasthen added. The slurry was stirred at room temperature for 30 minutesand filtered. The cake was washed with hexane (2×300 ml) and dried in avacuum-oven (30-35° C.) for 3 hours. The product, p-toluenesulfinic acid(240.0 g.), was obtained as a white powder.

Preparation 6N-[(2,5-DIFLUORO-PHENYL)-(TOLUENE-4-SULFONYL)-METHYL]-FORMAMIDE

[0198]

[0199] A 5 L three-necked round-bottomed flask, equipped with amechanical stirrer, a condenser and a thermometer, was charged with2,5-difluorobenzaldehyde (142.11 g, 1 mole). Toluene (500 ml),acetonitrile (500 ml), formamide (99.3 ml, 2.5 moles) andchlorotrimethylsilane (139.6 ml, 1.1 moles) were added respectively. Thecloudy mixture was heated to 50° C. and stirred at this temperature for7 hours. p-Toluenesulfinic acid (218.68 g, 1.4 moles) was added. Themixture was stirred at 50° C. for 6 hours and then 13 hours at roomtemperature. Methyl t-butyl ether (1.8 L) and water (1.7 L) were thenadded. The mixture was stirred at room temperature for 15 minutes atwhich time the organic layer was separated. The aqueous layer wasextracted with methyl t-butyl ether (500 ml). Most of the solvents wereremoved from the combined organic extracts. To the residual whitesemi-solid, hexane (1 L) and water (1 L) were added. The slurry wasstirred at room temperature for 30 minutes and filtered. The cake waswashed with hexane (2×200 ml) and dried in a vacuum-oven (30° C.) for 18hours. The product,N-[(2,5-Difluoro-phenyl)-(toluene-4-sulfonyl)-methyl]formamide (258.3 g,yield 79%,), was obtained as a white powder.

Preparation 7 [α-(P-TOLUENESULFONYL)-2,5-DIFLUOROBENZYL]ISONITRILE

[0200]

[0201] A 5 L three-necked round-bottomed flask, equipped with amechanical stirrer, an addition funnel and a thermometer, was chargedwith N-[(2,5-Difluoro-phenyl)-(toluene-4-sulfonyl)-methyl]-formamide(207.0 g, 0.636 moles) and tetrahydrofuran (J. T. Baker, low water, 1.5L). Phosphorous oxychloride (118.6 ml, 1.27 moles) was quickly pouredinto the reaction mixture (less than 5 minutes). The mixture was stirredat room temperature for 10 minutes and then cooled to 4° C. using anice/water bath. 2,6-Lutidine (445 ml, 3.82 moles) was added via theaddition funnel over a period of 30 minutes. The cooling bath was thenremoved and the mixture was stirred at room temperature for 18 hours.The reaction mixture was poured into a stirred and ice-water cooledsolution of 1.5 kg of ice and 1.1 L of saturated aqueous sodiumbicarbonate (NaHCO₃). The mixture was then extracted with ethyl acetate(2 L plus 1.5 L). The combined organic extracts were washed with 1 Naqueous hydrochloric acid (3 L), saturated aqueous NaHCO₃ (3 L) andbrine (3 L); and then dried (MgSO4). After removing all solvents,isopropanol (1.8 L) was added to the residual brownish solid. Theresulting slurry was stirred at room temperature for 2 hours. Water (0.9L) was added and the slurry was stirred for additional 30 minutes atroom temperature and then filtered. The cake was washed with 2:1isopropanol-water (2×500 ml) and dried in a vacuum-oven (30° C.) for 48hours. The product, [α-(p-Toluenesulfonyl)-2,5-difluorobenzyl]isonitrile(133.4 g, yield 68%,), was obtained as a brownish powder.

[0202] H¹ NMR (400 MHz, CDCl₃): δ, 7.7 (d, J=8.3 Hz, 2H) 7.41 (d, J=8.3Hz, 2H), 7.18 (m, 3H), 5.91 (s, 1H), 2.50 (s. 3H).

Preparation 8 [α-(P-TOLUENESULFONYL)-2,5-DIFLUOROBENZYL]ISONITRILE

[0203]

[0204] To a clean a dry nitrogen purged acetone boiled out 100 gallonglass lined reactor was charged, 7.9 Kg ofN-[(2,5-Difluoro-phenyl)-(toluene-4-sulfonyl)-methyl]-formamide (24,moles), 16 gallons of tetrahydrofuran and 7.8 Kg of phosphorousoxychloride (51 moles). The batch was allowed to stir at 20° C. for 30minutes and then cooled to 3.5° C. To the batch was added 15.8 Kg of2,6-lutidine (146 moles) over 15 minutes. The reaction mixture wasallowed to warm to 23° C. and was stirred for 17 hours at 23° C. Thereaction was judged complete by HPLC and was charged to a 40 gallonsolution of 10% sodium bicarbonate at 22° C., and the contents wereallowed to stir for 30 minutes. To the batch was then added 25 gallonsof ethyl acetate and the layers were separated. The water layer wasbackwashed with 9 gallons of ethyl acetate and the product rich ethylacetate combined with the first wash. The product rich ethyl acetatelayers were added to a 10% citric acid solution (20 gallons) and thenstirred. The organic layer was checked by HPLC for 2,6 lutidine and thenseparated. The organic layer was washed with 10 gallons of saturatedNaCl and dried over 7.9 Kg of magnesium sulfate. The drying agents wereremoved by filtration and the cake was washed with 4 gallons of ethylacetate. The ethyl acetate layer was concentrated to 7 gallons undervacuum at an internal temperature of 24° C. The batch was then added to11 gallons of IPO at 21° C. and allowed to granulate at 4° C. for 12hours. The product was isolated via filtration and washed with 4 gallonsof 5° C. IPO. The product was then dried at 34° C. for 22 hours withnitrogen bleed to recover 5.0 Kg of the title compound (66% yield).

Preparation 9 6-[OXAZOL-5-YL]-3-ISOPROPYL-[1,2,4]TRIAZOLO[4,3-a]PYRIDINE

[0205]

[0206] To a clean dry 5 liter round bottomed flask equipped with amechanical stirrer, nitrogen bubbler, heating mantle, temperaturecontroller, and condenser, was charged 3isopropyl-[1,2,4]triazolo(4,3-a)-6-pyridinecarboxaldehyde (140.9 grams,0.745 moles), potassium carbonate (133.8 grams, 0.968 moles),tosylmethyl isocyanide (146.9 grams, 0.745 moles), and methanol (2114ml). This mixture was heated at reflux and stirred for 1.5 to 2.0 hoursat 65 to 70° C. Assay by HPLC showed the reaction to be complete. Thepot was concentrated atmospherically to about one third of originalvolume. Water (1409 ml), was added and the pot further concentrated to apot temperature of 65 to 66° C. to remove the remaining methanol. Aftercooling, the desired product was extracted with methylene chloride (1409ml). The extraction was repeated twice with methylene chloride (2 times705 ml). The combined extracts were atmospherically concentrated anddisplaced with Isopropyl alcohol (420 ml). A thick slurry formed.Hexanes (1690 ml) were added and the slurry allowed to granulate for 12to 16 hours at 20 to 25° C. The solids were collected by vacuumfiltration, washed with hexanes, and dried to yield 111.45 grams, 97.8%purity (HPLC), 65.5% of theory.

[0207]¹H NMR (CDCl₃, 400 MHz) δ 8.23 (s, 1H), 7.98 (s, 1H), 7.82 (d, 1H,J=9.5 Hz), 7.467.43 (m, 2H), 3.43 (sept, 1H, J=7.05 Hz), 1.56 (d, 6H,J=7.05 Hz); MS 229 (M+1).

Preparation 106-[4-BROMO-OXAZOL-5-YL]-3-ISOPROPYL-[1,2,4]TRIAZOLO[4,3-a]PYRIDINE

[0208]

[0209] A clean, dry, 1 liter 4 neck round bottom flask equipped withmechanical stirrer, temperature probe, and purged with nitrogen, wascharged with 6-[oxazol-5-yl]-3-isopropyl[1,2,4]triazolo[4,3-a)pyridine(45.2 grams 0.198 moles) and dimethylformamide (271 ml). The pot wascooled below −60° C. with a dry icelacetone bath. Lithiumbis(trimethylsilyl)amide, 1 molar solution in tetrahydrofuran (198 ml0.198 moles), was added, keeping the temperature below −60° C. After theaddition was complete, the pot was further cooled to below −70° C. andstirred for 1 hour. While stirring, a solution of N-bromosuccinimide(35.24 g 0.198 moles) and dimethylformamide (105 ml), were stirred in aseparate 500 ml round bottom flask under nitrogen. After the one hourstir at −70° C., the solution of N-bromosuccinimide anddimethylformamide was slowly added to the anion keeping the temperaturebelow −70° C. After the addition, the reaction was continued for onehour below −70° C. The batch was then warmed to room temperature andquenched into methylene chloride (452 ml) and 1 N sodium hydroxide (452ml). The organic layer was then separated. The aqueous layer wasextracted a second time with methylene chloride (135 ml). The combinedorganic phase was washed with 1 N sodium hydroxide (452 ml) andsaturated brine solution (452 ml). The organic phase was then dried overmagnesium sulfate (50 grams) and concentrated/displaced with isopropylether (226 ml) to a temperature of 42° C. A thick slurry formed uponcooling. The solids were granulated at 20 to 25° C. for two hours,filtered, washed with isopropyl ether (50 ml), and dried to afford 53.0grams of light yellow solids, 96.4% purity (HPLC), 87% of theory.

[0210]¹H NMR (CDCl₃, 400 MHz) δ 8.56 (s, 1H), 7.95 (s, 1H), 7.85 (d, 1H,J=9.5 Hz), 7.77 (d, 1H, J=9.5 Hz), 3.43 (sept, 1H, J=7.05 Hz), 1.56 (d,6H, J=7.05 Hz); MS: 310, 309, 308, 307 (M⁺+1).

EXAMPLE 13-Cyclopropyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-1,2,4]triazolo[4,3-a]pyridine

[0211]

[0212] A) 5-Bromo-pyridine-2-yl-hydrazine

[0213] A mixture of 2,5-dibromopyridine (44.2 g, 0.187 moles), hydrazinehydrate (55% by weight, 105.7 mL, 1.87 mol), poly(ethylene glycol)(187.0 mL), 2-butanol (37.3 mL) and water (187.0 mL) under nitrogen isrefluxed gently for 29 hours. The mixture is cooled and stirred for 20hours. To the resulting slurry, cold water (220.0 mL) is added. Theslurry is stirred for an additional 30 minutes and filtered. The cake iswashed with cold water (3×) and dried in a vacuum-oven (40-45° C.) for48 hours. The title compound (30.5 g, 87%) may be obtained as off-whiteflakes.

[0214] B) 6-Bromo-3-cyclopropyl-[1,2,4]triazolo(4,3-a)pyridine

[0215] A mixture of 5-bromo-pyridin-2-yl-hydrazine (15.0 g, 79.8 mmol)and cyclopropane carbonyl chloride (65.0 mL, 71.8 mmol) is heated at 90°C. for 18 hours. The brown mixture is allowed to cool to roomtemperature, filtered, and washed with toluene to afford a light brownsolid. This solid is taken up in chloroform (CHCl₃), and washed withsaturated NaHCO₃. The organic layer is isolated, and the aqueous layerextracted twice with chloroform (CHCl₃). The combined organics arewashed with brine, dried over magnesium sulfate, and concentrated invacuo to give the title compound as a tan solid (18.2 g, 96%).

[0216] C) 3-Cyclopropyl-[1,2,4]triazolo(4.3-a)-6-pyridinecarboxaldehyde

[0217] To a cooled (−10° C.) solution of6-bromo-3-cyclopropyl-[1,2,4]triazolo(4,3-a)pyridine (4.8 g, 20.0 mmol)and THF (48.0 mL) is added a solution of isopropylmagnesium chloride inTHF (2.0M, 12.0 mL, 24.0 mmol) dropwise, maintaining the temperatureless than −5° C. The resulting slurry is stirred between 4 and 0° C. for30 minutes. DMF (3.9 mL, 50.0 mmol) is then added over 5 minutes. Thereaction is then heated at 50-55° C. for 2 hours, then cooled to roomtemperature. The reaction is poured into a cold solution of 10% citricacid. The reaction mixture is extracted with CHCl₃ (3×). The combinedorganics are washed with brine, dried over magnesium sulfate, andconcentrated in vacuo to a brownish solid (5.2 g). Silica gelchromatography, followed by ethyl acetate trituration yields the titlecompound as a yellow solid (1.8 g, 49%).

[0218] D) p-Toluenesulfinic Acid

[0219] A mixture of p-toluenesulfinic acid, sodium salt hydrate (39.2g), water (200.0 mL) and methyl t-butyl ether (MTBE, 200.0 mL) isstirred at room temperature for 10 minutes, then hydrochloric acid (37%wt. in water, 14.2 mL, 0.12 mol) is poured in over a period of 5minutes. The biphasic mixture is stirred at room temperature for 30minutes. The layers are separated and the aqueous layer extracted withMTBE (50.0 mL). The combined organic extracts are concentrated in vacuo(bath temperature below 35° C.) to a white semi-solid. Toluene (70.0 mL)is added to the residual solid. Most of solvents are removed and hexane(180.0 mL) is added. The slurry is stirred at room temperature for 30minutes and filtered. The cake is washed with hexane (2×) and dried in avacuum-oven (30-35° C.) for 3 hours. The product, p-toluenesulfinicacid, may be obtained as a white powder (24.0 g).

[0220] E)N-[(2.4-Difluoro-phenyl)-(toluene-4-sulfonyl)-methyl]-formamide

[0221] To 2,4-difluorobenzaldehyde (1.42 g, 10.0 mmol) is added toluene(5.0 mL), acetonitrile (5.0 mL), formamide (0.993 mL, 25.0 mmol) andchlorotrimethylsilane (1.40 mL, 11.0 mmol) in order. The cloudy mixtureis heated to 50° C. and stirred at this temperature for 7 hours.p-Toluenesulfinic acid (2.19 g, 14.0 mmol) is added, and the mixturestirred at 50° C. for 6 hours, then for 3 hours at room temperature.MTBE (18.0 mL) and water (17.0 mL) are added, and the mixture stirred atroom temperature for 15 minutes. The layers are separated, and theaqueous layer extracted with MTBE (5.0 mL). Most of the solvents areremoved from the combined organic extracts leaving a white semi-solid.To the residual is added hexane (10.0 mL) and water (10.0 mL), and theresulting slurry stirred at room temperature for 30 minutes, thenfiltered. The cake is washed with hexane (2×) and dried in a vacuum-oven(30° C.) for 18 hours. The title compound may be obtained as a whitepowder (2.58 g, 79%).

[0222] F) [α-(P-Toluenesulfonyl)-2,4-difluorobenzyl]isonitrile

[0223] To a mixture ofN-[(2,4-Difluoro-phenyl)-(toluene-4-sulfonyl)-methyl]-formamide (2.07 g,6.36 mmol) and THF (15 mL) is added phosphorous oxychloride (POCl₃)(1.19mL, 12.7 mmol) over a period of 5 minutes, and the resulting mixturestirred at room temperature for 10 minutes. The reaction is then cooledto 4° C. using an ice/water bath and 2,6-lutidine (4.45 mL, 38.2 mmol)is added over 30 minutes, maintaining the temperature less than 12° C.The cooling bath is removed and the mixture stirred at room temperaturefor 18 hours. The reaction mixture is poured into a stirred, ice watercooled, solution of ice and saturated aqueous NaHCO₃. The mixture isextracted with ethyl acetate (2×). The combined organic extracts arewashed with 1N aqueous hydrochloric acid, saturated aqueous NaHCO₃,brine and dried (MgSO₄). The solvents are removed in vacuo, andisopropanol (18.0 mL) added to the residual brownish solid. Theresulting slurry is stirred at room temperature for 2 hours, then water(9.0 mL) is added and the slurry stirred for an additional 30 minutes atroom temperature. The slurry is filtered, the cake washed with 2:1isopropanol-water (2×) and dried in a vacuum-oven (30° C.) for 48 hours.The title compound may be obtained as a tan solid (1.33 g, 68%).

[0224] G)3-Cyclopropyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine

[0225] A mixture of [α-(p-Toluenesulfonyl)-2,4-difluorobenzyl]isonitrile(123.0 mg, 0.40 mmol),3-cyclopropyl-[1,2,4]triazolo(4,3-a)-6-pyridinecarboxaldehyde (75.0 mg,0.40 mmol), potassium carbonate (66.0 mg, 0.48 mmol) and acetonitrile(2.0 mL) was heated at 70° C. for 22 hours. The reaction mixture wascooled to room temperature and poured into ice water and brine. Theaqueous layer was extracted with CHCl₃ (3×). The combined organics werewashed with brine, dried over magnesium sulfate, and concentrated invacuo to a yellow solid. Silica gel chromatography, followed byrecrystallization (Ethyl acetate) yielded a white solid (24.0 mg, 18%).LCMS (m/z) 339 (M+1). ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 8.06 (s,1H), 7.77 (d, 1H, J=10.4 Hz), 7.63-7.69 (m, 1H), 7.28-7.32 (m, 1H),7.03-7.08 (m, 1H), 6.91-6.96 (m, 1H), 1.96-2.03 (m, 1H), 1.15-1.24 (m,4H).

EXAMPLE 23-Cyclopropyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine

[0226]

[0227] This compound was prepared in an analogous manner to Example 1starting with 2,5-difluorobenzaldehyde in Step E. LCMS (m/z) 339 (M+1).

EXAMPLE 36-[4-(2,5-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine

[0228]

[0229] This compound was prepared in an analogous manner to Example 1,staring with 1-methylcyclopropane carbonyl chloride (synthesized fromcommercial 1-methylcyclopropane carboxylic acid) in Step B and2,5-difluorobenzaldehyde in Step E. LCMS (m/z) 353 (M+1).

EXAMPLE 46-[4-(2,4-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine

[0230]

[0231] This compound was prepared in an analogous manner to Example 1,starting with 1-methylcyclopropane carbonyl chloride (synthesized fromcommercial 1-methylcyclopropane carboxylic acid) in Step B. LCMS (m/z)353 (M+1).

EXAMPLE 53-Cyclobutyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine

[0232]

[0233] This compound was prepared in an analogous manner to Example 1,starting with cyclobutane carbonyl chloride in Step B and2,5-difluorobenzaldehyde in Step E. LCMS (m/z) 353 (M+1).

1. A compound of the formula

wherein R¹ is fluoro; s is two; R² is (C₃-C₆)cycloalkyl optionallysubstituted by one or two moieties independently selected from the groupconsisting of halo, (C₁-C₄)alkyl, hydroxy, (C₁-C₆)alkoxy, and(C₃-C₆)alkyl-(C═O)—O—; or a pharmaceutically acceptable salt thereof. 2.A compound according to claim 1 wherein R² is optionally substituted(C₃-C₆)cycloalkyl.
 3. A compound according to claim 2 wherein R² isoptionally substituted cyclopropyl or cyclobutyl.
 4. A compoundaccording to claim 2, wherein the compound has the formula


5. A compound according to claim 2, wherein the compound has the formula


6. A compound according to claim 1, wherein R² is (C₃-C₆)cycloalkyl. 7.A compound according to claim 2, wherein R² is (C₃-C₆)cycloalkylsubstituted with one or two (C₁-C₃)alkyl.
 8. A compound according toclaim 2, wherein R² is (C₃-C₆)cycloalkyl substituted with one or twomethyl groups.
 9. A compound according to claim 2, wherein R² is(C₃-C₆)cycloalkyl substituted with one (C₁-C₃)alkyl.
 10. A compoundaccording to claim 2, wherein R² is (C₃-C₆)cycloalkyl substituted withone methyl, ethyl or propyl group.
 11. A compound according to claim 2,wherein said compound is selected from the group consisting of:3-Cyclobutyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;6-[4-(2,4-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;6-[4-(2,5-Difluoro-phenyl)-oxazol-5-yl]-3-(1-methyl-cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridine;3-Cyclopropyl-6-[4-(2,5-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine;and3-Cyclopropyl-6-[4-(2,4-difluoro-phenyl)-oxazol-5-yl]-[1,2,4]triazolo[4,3-a]pyridine.12. A method of treating an MAP kinase mediated disease in a mammal inneed thereof, which comprises administering to said mammal an effectiveamount of a compound according to claim
 1. 13. A method of treating ap38 kinase mediated disease in a mammal in need thereof, which comprisesadministering to said mammal an effective amount of a compound accordingto claim
 1. 14. A method for treating a condition selected from thegroup consisting of arthritis, psoriatic arthritis, Reiter's syndrome,rheumatoid arthritis, gout, traumatic arthritis, rubella arthritis andacute synovitis, rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, gouty arthritis and other arthritic condition, sepsis,septic shock, endotoxic shock, gram negative sepsis, toxic shocksyndrome, Alzheimer's disease, stroke, neurotrauma, asthma, adultrespiratory distress syndrome, cerebral malaria, chronic pulmonaryinflammatory disease, silicosis, pulmonary sarcoidosis, bone resorptiondisease, osteoporosis, restenosis, cardiac and renal reperfusion injury,thrombosis, glomerularonephritis, diabetes, graft vs. host reaction,allograft rejection, inflammatory bowel disease, Crohn's disease,ulcerative colitis, multiple sclerosis, muscle degeneration, eczema,contact dermatitis, psoriasis, sunburn, and conjunctivitis shock in amammal, including a human, comprising administering to said mammal anamount of a compound according to claim 1 effective in treating such acondition.
 15. A pharmaceutical composition for the treatment of acondition selected from the group consisting of arthritis, psoriaticarthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumaticarthritis, rubella arthritis and acute synovitis, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic condition, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, Alzheimer's disease, stroke,neurotrauma, asthma, adult respiratory distress syndrome, cerebralmalaria, chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, bone resorption disease, osteoporosis, restenosis, cardiacand renal reperfusion injury, thrombosis, glomerularonephritis,diabetes, graft vs. host reaction, allograft rejection, inflammatorybowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis,muscle degeneration, eczema, contact dermatitis, psoriasis, sunburn, andconjunctivitis shock in a mammal, including a human, comprising anamount of a compound of claim 1 effective in such treatment and apharmaceutically acceptable carrier.
 16. A pharmaceutical compositionfor the treatment of a condition which can be treated by the inhibitionof MAP kinase in a mammal, including a human, comprising an amount of acompound of claim 1 effective in such treatment and a pharmaceuticallyacceptable carrier.
 17. A pharmaceutical composition for the treatmentof a condition which can be treated by the inhibition of p38 kinase in amammal, including a human, comprising an amount of a compound of claim 1effective in such treatment and a pharmaceutically acceptable carrier.